Substituted pyrrolidine derivative

ABSTRACT

A quinolone antibacterial compound, or a salt or hydrate of the compound, for the treatment of infectious diseases, which exhibit potent antibacterial activity and higher selective toxicity against Gram-positive and Gram-negative bacteria, which do not cause side effects (e.g., convulsion), which exhibit higher safety, and which has a structure of formula (I):

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 11/596,318,filed on Nov. 13, 2006, which is a National Stage (371) ofPCT/JP05/08750, filed on May 13, 2005, which claims priority to JP2004-143352, filed on May 13, 2004.

TECHNICAL FIELD

The present invention relates to a synthetic quinolone antibacterialdrug which is useful as a drug, an veterinary drug, a fishery drug, oran antibacterial preservative; and more particularly to a syntheticquinolone antibacterial drug containing a1,4-dihydro-4-oxoquinoline-3-carboxylic acid (hereinafter abbreviated as“quinolone”) skeleton having, at position 7, a3-amino-4-aliphatic-hydrocarbon-group-substituted-pyrrolidin-1-yl group.The term “synthetic quinolone antibacterial drug” encompasses asynthetic quinolone antibacterial drug containing merely a quinoloneskeleton, and a synthetic quinolone antibacterial drug containing, as abasic skeleton, a quinolone-based skeleton further having a condensedring, as in the case of2,3-dihydro-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid(hereinafter abbreviated as “pyridobenzoxazine”).

BACKGROUND ART

Since the discovery of norfloxacin, synthetic quinolone antibacterialdrugs have been improved in their antibacterial activity andpharmacokinetics, and many compounds have been employed in the clinicalfield as chemotherapeutic agents which are effective for almost allsystemic infectious diseases.

However, in recent years, bacteria exhibiting low sensitivity tosynthetic quinolone antibacterial drugs have become increasingly commonin the clinical field. For example, there has been an increase inGram-positive bacteria which exhibit resistance to drugs other thansynthetic quinolone antibacterial drugs and exhibit low sensitivity tosynthetic quinolone antibacterial drugs, including β-lactam-insensitiveGram-positive cocci such as Staphylococcus aureus (MRSA) andStreptococcus neumoniae (PRSP), andaminoglycoside-antibacterial-drug-insensitive Enterococcus (VRE).Therefore, particularly in the clinical field, demand has arisen for adrug exhibiting higher efficacy on Gram-positive cocci.

As has been shown, when administered in combination with nonsteroidalanti-inflammatory drugs (NSAIDs), some synthetic quinolone antibacterialdrugs cause side effects, including convulsion, central actions (e.g.,mild central nervous disorders such as stagger, headache, and insomnia,and severe side effects such as convulsion), phototoxicity(photosensitivity), hepatotoxicity, cardiotoxicity(fatal-arrhythmia-inducing abnormality which is observed as abnormalelectrocardiogram), and abnormal blood glucose level. Therefore, demandhas arisen for development of a synthetic quinolone antibacterial drugexhibiting higher safety (see, for example, Non-Patent Document 1).

As has been known, the antibacterial activity, pharmacokinetics, andsafety of a synthetic quinolone antibacterial drug are greatly affectedby the structure of a substituent which is present at position 7 (or aposition corresponding thereto) of the quinolone skeleton. Quinolonederivatives in which the quinolone skeleton is substituted, at position7, with a 3-amino-4-methylpyrrolidin-1-yl group have been known toexhibit potent antibacterial activity to Gram-negative and Gram-positivebacteria (see, for example, Non-Patent Documents 2 and 3).

However, most of the aforementioned3-amino-4-methylpyrrolidin-1-yl-group-substituted quinolone derivativesexhibit high incidence of chromosomal aberration, potent cytotoxicity,potent mouse or rat bone marrow micronucleus induction, and lowselective toxicity, as compared with the case of quinolone derivativeshaving, as a substituent, a substituted or non-substituted piperazinylgroup. Therefore, such a3-amino-4-methylpyrrolidin-1-yl-group-substituted quinolone derivativeacts on bacteria and as well on eukaryotic cells (see Non-PatentDocument 3), and thus is difficult to employ as a drug or a veterinarydrug. In practice, such a quinolone derivative has not yet been employedclinically.

Known quinolone derivatives substituted at position 7 with a3-amino-4-alkylpyrrolidin-1-yl-group, which are related to the compoundof the present invention, are described below (see Patent Documents 1and 2).

The substituents of a compound of this formula are defined in PatentDocument 1, etc. Although the substituents may be denoted by symbolsthat are common to those as used herein, the definitions provided in theprior art should be considered as irrelevant to those of the presentinvention. Specifically disclosed is merely a compound in which thesubstituent at position 4 of a pyrrolidinyl group (corresponding to thegroup R²) is a methyl group.

Patent Documents 3 and 4 disclose a compound having the followingstructure; specifically, merely a compound in which the substituent atposition 4 of a pyrrolidinyl group is a methyl group (the substituentsof this compound are defined in Patent Document 3, etc., and thus, evenwhen the substituents are represented by the same symbols as usedherein, they are irrelevant to those as defined herein).

Patent Documents 3 and 4 also disclose a compound having the followingpyridobenzoxazine skeleton. The compound, in which the substituent atposition 7 is a cis-3-amino-4-methylpyrrolidin-1-yl group, is adiastereomeric mixture.

Patent Document 5 discloses a compound having the following structure,in which the substituent at position 4 of a pyrrolidinyl group(corresponding to the group R²) is a methyl group, and the substituentat position 1 of the quinolone skeleton (corresponding to the group R¹)is a 2-fluorocyclopropyl group (the substituents of the compounddisclosed in Patent Document 5 are defined in Patent Document 5, andthus, even when the substituents are represented by the same symbols asused herein, they are irrelevant to those as defined herein). Accordingto this patent document, the 1-position substituent is preferably anon-substituted cyclopropyl group.

Patent Document 6 does not disclose a compound in which the 1-positionsubstituent is a halogenocyclopropyl group.

Patent Document 7 discloses a compound in which the substituent atposition 4 of a pyrrolidinyl group is an ethyl group, but does notdescribe specific examples of the compound. In addition, the substituentat position 1 of the quinolone skeleton is limited to a non-substitutedcyclopropyl group.

Patent Documents 8 and 9 disclose a compound in which the substituent atposition 4 of a pyrrolidinyl group (corresponding to the group R¹) is anethyl group (the substituents of the compound disclosed in PatentDocuments 8 and 9 are defined in Patent Document 8, etc., and thus, evenwhen the substituents are represented by the same symbols as usedherein, they are irrelevant to those as defined herein). However, thesubstituent at position 1 of the quinolone skeleton is a non-substitutedcyclopropyl group.

Patent Documents 10 and 11 disclose a compound having the followingstructure.

-   Patent Document 1: Specification of JP Patent No. 2917010-   Patent Document 2: Specification of U.S. Pat. No. 5,587,386-   Patent Document 3: Specification of European Patent No. 208210-   Patent Document 4: Specification of U.S. Pat. No. 4,753,953-   Patent Document 5: JP-A-SHO63-264461-   Patent Document 6: U.S. Pat. No. 4,855,292-   Patent Document 7: JP-A-SHO64-83068-   Patent Document 8: WO 96/22988 pamphlet-   Patent Document 9: JP-A-HEI8-259561-   Patent Document 10: Specification of European Patent No. 242789-   Patent Document 11: Specification of U.S. Pat. No. 4,886,810-   Non-Patent Document 1: Clinical Application of New Quinolone Agent,    edited by Hiroyuki Kobayashi, Iyaku Journal Co., Ltd. (2001)-   Non-Patent Document 2: International Journal of Antimicrobial    Agents, Vol. 16, p. 5 (2000)-   Non-Patent Document 3: Journal of Antimicrobial Chemotherapy, Vol.    33, p. 685 (1994)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Therefore, the present invention contemplates provision of a quinoloneantibacterial agent and a drug for the treatment of infectious diseases(hereinafter may be referred to as an “infectious disease treatingdrug”), which exhibit potent antibacterial activity and higher selectivetoxicity against Gram-positive and Gram-negative bacteria, which do notcause side effects (e.g., convulsion), and which exhibit higher safety.

Means for Solving the Problems

In view of the foregoing, the present inventors have conducted extensivestudies, and as a result, have found that a compound represented by thefollowing formula (I), a salt of the compound, or a hydrate of thecompound or the salt exhibits antibacterial activity againstGram-positive and Gram-negative bacteria at a level almost comparable tothat of a known synthetic quinolone antibacterial drug, and exhibitssuch a higher safety that it can be employed as an antibacterial drug oran infectious disease treating drug. The present invention has beenaccomplished on the basis of these findings.

Accordingly, the present invention provides a compound represented bythe following formula (I):

[wherein R¹ represents a hydrogen atom, a C1-C6 alkyl group, or asubstituted carbonyl group derived from an amino acid, a dipeptide, or atripeptide;

-   R² represents a hydrogen atom or a C1-C6 alkyl group, and, when    either one or each of R¹ and R² is an alkyl group, the alkyl group    may be substituted with a group selected from the group consisting    of a hydroxyl group, an amino group, a halogen atom, a C1-C6    alkylthio group, and a C1-C6 alkoxy group;-   R² represents a C2-C6 alkyl group, a C2-C6 alkenyl group, or a C3-C6    cycloalkyl group;-   R⁴ and R⁵ each independently represents a hydrogen atom or a C1-C6    alkyl group;-   R⁶ represents a C3-C6 halogenocycloalkyl group;-   R⁷ represents a hydrogen atom, a phenyl group, an acetoxymethyl    group, a pivaloyloxymethyl group, an ethoxycarbonyl group, a choline    group, a dimethylaminoethyl group, a 5-indanyl group, a phthalidyl    group, a 5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl group, a    3-acetoxy-2-oxobutyl group, a C1-C6 alkyl group, a C2-C7    alkoxymethyl group, or a phenylalkyl group formed of a C1-C6    alkylene group and a phenyl group;-   X¹ represents a hydrogen atom or a halogen atom; and-   A represents a nitrogen atom or a partial structure represented by    the following formula (II):

(wherein X² represents a C1-C6 alkyl group or a C1-C6 alkoxy group; X²and the aforementioned R⁶ may together form a cyclic structure so as tocontain a part of the quinolone nucleus; the thus-formed ring maycontain, as a ring-constituting atom, an oxygen atom, a nitrogen atom,or a sulfur atom; and the ring may be substituted with a C1-C6 alkylgroup which may have a substituent)];

-   a salt, or a hydrate thereof.

The present invention also provides7-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-(S)-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylicacid, a salt, or a hydrate thereof;7-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylicacid, a salt, or a hydrate thereof; and(3S)-10-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid, a salt, or a hydrate thereof.

The present invention also provides a drug, an antibacterial drug, andan infectious disease treating drug, each of the drugs containing, as anactive ingredient, a compound represented by formula (I), a salt, or ahydrate thereof.

The present invention also provides a method for treating a pathologicalcondition, characterized by comprising administration of an effectiveamount of a compound represented by formula (I), a salt, or a hydratethereof; and a method for treating an infectious disease, characterizedby comprising administration of an effective amount of a compoundrepresented by formula (I), a salt, or a hydrate thereof.

The present invention also provides a method for producing a drug,characterized by comprising incorporation of a compound represented byformula (I), a salt, or a hydrate thereof; a method for producing anantibacterial drug, characterized by comprising incorporation of acompound represented by formula (I), a salt, or a hydrate thereof; and amethod for producing an infectious disease treatment drug, characterizedby comprising incorporation of a compound represented by formula (I), asalt, or a hydrate thereof.

The present invention also provides use of a compound represented byformula (I), a salt, or a hydrate thereof for producing a drug; use of acompound represented by formula (I), a salt, or a hydrate thereof forproducing an antibacterial drug; and use of a compound represented byformula (I), a salt, or a hydrate thereof for producing an infectiousdisease treating drug.

Effects of the Invention

The substituted pyrrolidine derivative of the present invention exhibitslow convulsion-inducing effect and chromosomal-aberration-inducingeffect, and higher safety, although the antibacterial activity of thederivative against Gram-positive and Gram-negative bacteria is almostcomparable to that of a known synthetic quinolone antibacterial drug.Therefore, the substituted pyrrolidine derivative of the presentinvention is useful as an antibacterial drug or an infectious diseasetreatment drug.

BEST MODE FOR CARRYING OUT THE INVENTION

Now will be described the substituents of a compound of the presentinvention represented by formula (I).

R¹ represents a hydrogen atom, a C1-C6 alkyl group, or a substitutedcarbonyl group derived from an amino acid, dipeptide, or a tripeptide.R² represents a hydrogen atom or a C1-C6 alkyl group. Examples of theC1-C6 alkyl group represented by R¹ or R² include linear alkyl groupssuch as methyl, ethyl, n-propyl, n-butyl, and n-pentyl; and branchedalkyl groups such as isopropyl, isobutyl, sec-butyl, and tert-butyl.Preferably, each of R¹ and R² is a hydrogen atom; or one of R¹ and R² isa hydrogen atom, and the other is a C1-C6 alkyl group. Particularlypreferably, each of R¹ and R² is a hydrogen atom; or one of R¹ and R² isa hydrogen atom, and the other is a methyl group.

A compound of formula (I) in which R¹ is a substituted carbonyl groupderived from an amino acid, a dipeptide, or a tripeptide is useful as aprodrug. Examples of substituents employed for producing such a prodruginclude a substituted carbonyl group derived from an amino acid (e.g.,glycine, alanine, or aspartic acid), a dipeptide (e.g., glycine-glycine,glycine-alanine, or alanine-alanine), or a tripeptide (e.g.,glycine-glycine-alanine or glycine-alanine-alanine).

When either one or each of R¹ and R² is an alkyl group, the alkyl groupmay be substituted with a group selected from the group consisting of ahydroxyl group, an amino group, halogen atom, a C1-C6 alkylthio group,and a C1-C6 alkoxy group. When the alkyl group is substituted with ahydroxyl group or an amino group, more preferably, such a substituent ispresent on the terminal carbon atom of the alkyl group. Preferredhydroxyl-group-containing alkyl groups include hydroxyalkyl groupshaving three or less carbon atoms, such as a hydroxymethyl group, a2-hydroxyethyl group, a 2-hydroxypropyl group, and a 3-hydroxypropylgroup. Preferred amino-group-containing alkyl groups include aminoalkylgroups having three or less carbon atoms, such as an aminomethyl group,a 2-aminoethyl group, a 2-aminopropyl group, and a 3-aminopropyl group.

When the alkyl group is substituted with a halogen atom, the alkyl groupmay be any of C1-C6 linear and branched alkyl groups, and the halogenatom is preferably a fluorine atom. No particular limitation is imposedon the number of fluorine atom(s), and such a fluoroalkyl group may be amonofluoroalkyl group to a perfluoroalkyl group. Examples of thefluorine-substituted alkyl group include a monofluoromethyl group, adifluoromethyl group, a trifluoromethyl group, and a2,2,2-trifluoroethyl group.

When the alkyl group is substituted with an alkylthio group or an alkoxygroup, the alkyl group may be a linear or branched alkyl group, and thealkyl group constituting the alkylthio or alkoxy group may be a linearor branched alkyl group. The alkylthio-group-containing alkyl group ispreferably an alkylthiomethyl group, an alkylthioethyl group, or analkylthiopropyl group, and the alkylthio group is preferably a C1-C3alkylthio group. More preferred alkylthio-group-containing alkyl groupsinclude a methylthiomethyl group, an ethylthiomethyl group, and amethylthioethyl group. The alkoxy-group-containing alkyl group ispreferably an alkoxymethyl group, an alkoxyethyl group, or analkoxypropyl group, and the alkoxy group is preferably a C1-C3 alkoxygroup. More preferred alkoxy-group-containing alkyl groups include amethoxymethyl group, an ethoxymethyl group, and a methoxyethyl group.

Examples of the C2-C6 alkyl group represented by R³ include linear alkylgroups such as ethyl, n-propyl, n-butyl, and n-pentyl; and branchedalkyl groups such as isopropyl, isobutyl, sec-butyl, and tert-butyl. Anethyl group, an n-propyl group, or an isopropyl group is preferred, withan ethyl group being particularly preferred. Examples of the C3-C6cycloalkyl group include a cyclopropyl group, a cyclobutyl group, acyclopentyl group, and a cyclohexyl group. A cyclopropyl group or acyclobutyl group is preferred, with a cyclopropyl group beingparticularly preferred. Examples of the C2-C6 alkenyl group include avinyl group, a 1-propenyl group, a 2-propenyl group, and an isopropenylgroup. A vinyl group or an isopropenyl group is preferred, with a vinylgroup being particularly preferred. Of these substituents represented byR³, an ethyl group is particularly preferred.

R⁴ and R⁵ each independently represents a hydrogen atom or a C1-C6 alkylgroup. Preferably, each of R⁴ and R⁵ is a hydrogen atom. The C1-C6 alkylgroup may be a C1-C6 alkyl group similar to that described above.

A C3-C6 halogenocycloalkyl group, which is represented by R⁶, refers toany of the aforementioned cycloalkyl groups substituted with one or twohalogen atoms (e.g., fluorine, chlorine, and bromine). Such a cycloalkylgroup is preferably a monohalogenocyclopropyl group or adihalogenocyclopropyl group, particularly preferably amonofluorocyclopropyl group.

Examples of the substituent represented by R⁷ include a hydrogen atom, aphenyl group, an acetoxymethyl group, a pivaloyloxymethyl group, anethoxycarbonyl group, a choline group, a dimethylaminoethyl group, a5-indanyl group, a phthalidyl group, a5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl group, a 3-acetoxy-2-oxobutyl group,any of the aforementioned C1-C6 alkyl groups, a C2-C7 alkoxymethylgroup, and a phenylalkyl group formed of a C1-C6 alkylene group and aphenyl group. A C2-C7 alkoxymethyl group refers to a methyl groupsubstituted with any of the aforementioned C1-C6 alkoxy groups. Specificexamples of the C2-C7 alkoxymethyl group include a methoxymethyl group,an ethoxymethyl group, and a propoxymethyl group. Specific examples ofthe phenylalkyl group formed of a C1-C6 alkylene group and a phenylgroup include a phenylmethyl group and a phenethyl group. Of thesesubstituents represented by R⁷, a hydrogen atom is particularlypreferred.

A quinolone carboxylic acid derivative in which the carboxylic moiety isesterified is useful as a synthetic intermediate or a prodrug. Examplesof the ester useful as a synthetic intermediate include an alkyl ester,a benzyl ester, an alkoxyalkyl ester, a phenylalkyl ester, and a phenylester. Examples of the ester useful as a prodrug (i.e., an ester whichis readily cleaved in vivo to form a free carboxylic acid) include anacetoxymethyl ester, a pivaloyloxymethyl ester, an ethoxycarbonyl ester,a choline ester, a dimethylaminoethyl ester, a 5-indanyl ester, aphthalidinyl ester, a 5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl ester, and a3-acetoxy-2-oxobutyl ester.

X¹ represents a hydrogen atom or a halogen atom. A hydrogen atom or afluorine atom is preferred, with a fluorine atom being particularlypreferred.

In the partial structure represented by A, X² represents a C1-C6 alkylgroup or a C1-C6 alkoxy group. The C1-C6 alkyl group may be any of theaforementioned C1-C6 alkyl groups. The C1-C6 alkoxy group may be analkoxy group derived from the C1-C6 alkyl group. Of these substituents,a C1-C3 alkyl group or a C1-C3 alkoxy group is preferred, with a methylgroup or a methoxy group being particularly preferred. The partialstructure may be a cyclic structure which is formed by X² and R⁶ so asto contain a part of the quinolone nucleus. The thus-formed ring ispreferably a 5- to 7-membered ring, particularly preferably a 6-memberedring. The thus-formed ring may be saturated or unsaturated. The cyclicstructure may contain, as a ring-constituting atom, an oxygen atom, anitrogen atom, or a sulfur atom, and may be substituted with any of theaforementioned C1-C6 alkyl groups. Preferably, the thus-formed cyclicstructure contains an oxygen atom, and is substituted with a methylgroup. The partial structure which forms the cyclic structure ispreferably a structure represented by the formula: —O—CH₂—CH(—CH₃)— (thecarbon atom on the extreme right is bonded to the nitrogen atom of thequinolone skeleton).

The compound (I) of the present invention having such a partialstructure preferably has, as a basic skeleton, a pyridobenzoxazineskeleton represented by the following formula (A):

[In formula A, the arrow denotes a bond to be connected with theaforementioned pyrrolidine ring, and X¹ and R⁷ have the same meanings asdefined above.]

In the compound of the present invention represented by formula (I), thesubstituent at position 7 (or the substituent at position 10 in the casewhere the compound has a pyridobenzoxazine skeleton); i.e., a3-amino-4-aliphatic-group-substituted-pyrrolidin-1-yl group isrepresented by the following formula (B).

Therefore, the compound has four optical isomers based on the asymmetriccarbons at positions 3 and 4 of this substituent. Of these opticalisomers, 3,4-cis isomers are preferred, and a (3S,4S)-configuration or(3S,4R)-configuration isomer is more preferred, with a(3S,4S)-configuration isomer represented by the following formula (B1)being particularly preferred.

[In formulas (B) and (B1), R¹, R², R³, R⁴ and R⁵ have the same meaningsas defined above.]

The stereochemistry of the halogenocyclopropyl group represented by R⁶is preferably such that the halogen atom and the quinolone carboxylicacid skeleton are in a 1,2-cis configuration with respect to thecyclopropane ring. As used herein, the term “cis configuration” refersto the case where the halogen atom and the quinolone carboxylic acidskeleton are in a cis configuration with respect to the cyclopropanering. The cis configuration may be a (1R,2S)-configuration or a(1S,2R)-configuration, but the former is preferred.

When the compound of the present invention represented by formula (I),which has such a structure that produces diastereomers, is to beadministered to an animal or a human, preferably, a compound containinga single diastereomer is selected for administration. The expression “acompound containing a single diastereomer” refers to the case where thecompound does not contain an additional diastereomer at all, and as wellthe case where the compound contains an additional diastereomer to suchan extent that the additional diastereomer does not affect the physicalconstant and activity of the compound. As used herein, the expression“stereochemically single” refers to the case where, when a compound hasoptical isomers, the compound is formed of a single optical isomer, orthe compound contains an additional optical isomer to such an extentthat the additional optical isomer does not affect the physical constantand activity of the compound. The compound (I) of the present inventionis preferably a compound in which the substituents at positions 3 and 4of the 7-position substituent are in a (3S,4S)-configuration, and thehalogen atom and the quinolone carboxylic acid skeleton are in a(1R,2S)-configuration or a (3S,4S)-configuration with respect to thecyclopropane ring of the halogenocyclopropyl group represented by R⁶. Inview that the aforementioned pyridobenzoxazine skeleton contains aquinolone skeleton, a compound having the pyridobenzoxazine skeleton isparticularly preferred.

The compound (I) of the present invention may be in a free form, or maybe in the form of an acid addition salt or a carboxylic salt. Examplesof the acid addition salt include inorganic acid salts such ashydrochloride, sulfate, nitrate, hydrobromide, hydroiodide, andphosphate; and organic acid salts such as sulfonates (e.g.,methanesulfonate, benzenesulfonate, and p-toluenesulfonate) andcarboxylates (e.g., acetate, citrate, maleate, fumarate, and lactate).Examples of the carboxylic salt include alkali metal salts such aslithium salt, sodium salt, and potassium salt; alkaline earth metalsalts such as magnesium salt and calcium salt; ammonium salt;triethylamine salt; N-methylglucamine salt; andtris-(hydroxymethyl)aminomethane salt. The free form, acid additionsalt, or carboxylic salt of the compound (I) of the present inventionmay be present in the form of a hydrate.

Specific examples of the compound (I) of the present invention include:

-   7-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-(S)-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 1);-   7-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 2);-   (3S)-10-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 3);-   7-[(3S,4S)-3-amino-4-propylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 4);-   7-[(3S,4S)-3-amino-4-propylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 5);-   (3S)-10-[(3S,4S)-3-amino-4-propylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 6);-   (3S)-10-[(3S,4S)-3-amino-4-vinyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 7);-   7-[(3S,4S)-3-amino-4-cyclopropylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 8);-   (3S)-10-[(3S,4S)-3-amino-4-cyclopropylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 9);-   (3S)-10-[(3S)-3-amino-4-isopropyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 10);-   (3S)-10-[(3S,4R)-3-amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 11);-   7-[(3S,4S)-3-ethyl-4-methylaminopyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 12);-   (3S)-10-[(3S,4S)-3-ethyl-4-methylaminopyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 13);-   7-[(3S,4S)-3-amino-4-ethylpyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 14);-   7-[(3S,4S)-3-ethyl-4-methylaminopyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 15);-   7-[(3S,4S)-3-amino-4-isopropylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 16); and-   6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-7-[(3S,4S)-3-isopropyl-4-methylaminopyrrolidin-1-yl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, a salt, or a hydrate thereof (Compound No. 17).

Of these, particularly preferred are7-[(3S,4R)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-(S)-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylicacid, a salt, or a hydrate thereof (Compound No. 1);7-[(3S,4R)-3-amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylicacid, a salt, or a hydrate thereof (Compound No. 2); or(3S)-10-[(3S,4R)-3-amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid, a salt, or a hydrate thereof (Compound No. 3).

The compound of the present invention represented by formula (I) can beproduced through a variety of methods. In a preferred production method,the compound is produced, for example, by reacting a compoundrepresented by the following formula (VI-1) or (VI-2):

[wherein R⁶, R⁷, X¹ and A have the same meanings as defined above; X²represents a leaving group; and Q represents a C1-C6 alkyl group]

-   or a boron chelate obtained through conversion of the —COOR⁷ moiety    of this compound into —COOBF₂, —COOB(OAc)₂, or the like with a    3-amino-4-aliphatic-substituted-pyrrolidine derivative represented    by the following formula (VII):

[R¹, R², R³, R⁴ and R⁵ have the same meanings as defined above]

-   or an addition salt of the derivative. A compound represented by    formula (VII) may be prepared upon use; i.e., the compound (VII) may    be prepared, from a compound in which the nitrogen on the ring is    protected, by removing the protective group during the course of    reaction. Examples of the leaving group X² include substituted    sulfonyloxy groups such as methanesulfonyloxy,    trifluoromethanesulfonyloxy, benzenesulfonyloxy, and    toluenesulfonyloxy; and halogen atoms. The addition salt may be any    of the aforementioned addition salts. The production method is    described in detail in, for example, WO 02/40478 pamphlet or    specification of Japanese Patent Application No. 2003-336864. When a    boron chelate is employed for reaction, the boron chelate may be    produced through a known method. Reaction between a boron chelate    and a compound of formula (VII) may be carried out in the presence    of a base. After completion of reaction, the boron chelate moiety    may be cleaved through hydrolysis in the presence of a base.    Specifically, these steps may be performed through a known method.

Next will be described typical methods for producing3-amino-4-aliphatic-substituted-pyrrolidine derivatives (in particular,(3S,4S)-3-amino-4-aliphatic-substituted-pyrrolidine derivatives).However, the pyrrolidine derivative production method is not limited tothe below-described methods.

[In the reaction scheme, “Boc” denotes a tert-butoxycarbonyl group;“Cbz” denotes a benzyloxycarbonyl group; X³ represents a leaving groupfor nucleophilic substitution; and R⁸ has the same meaning as definedabove in R³.]

In step 1, a leaving group for nucleophilic substitution (step 2) isintroduced into the hydroxymethyl moiety of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-hydroxymethylpyrrolidine.(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-hydroxymethylpyrrolidinecan be produced through the method described in WO 99/65918 pamphlet.Examples of the leaving group include substituted sulfonyloxy groups. Amethanesulfonyloxy group, a trifluoromethanesulfonyloxy group, abenzenesulfonyloxy group, or a p-toluenesulfonyloxy group is preferred,with a p-toluenesulfonyloxy group being particularly preferred. Theleaving group may be a halogen atom. The halogen atom is preferably abromine atom or an iodine atom. Introduction of a substitutedsulfonyloxy group or halogenation can be performed under generallyemployed conditions.

In step 2, a carbon-carbon bond is formed through nucleophilicsubstitution. Examples of the reaction reagent to be employed includeorganic alkyllithium reagents, which are generally employed for couplingreaction with an alkyl halide; Grignard reagents; and organocopperreagents employed for S_(N)2 substitution, such as Gilman reagents(e.g., dialkyl copper lithium-lithium halide and dialkyl copperlithium-Lewis acid complex). Of these, methyllithium is preferred.Generally, the reaction can be performed by use of a solvent which isinert to organic metal (e.g., an ether solvent such as diethyl ether ortetrahydrofuran). The reagent to be employed may be a commerciallyavailable one, or may be prepared upon use or in the reaction system.

In step 3, no particular limitation is imposed on the deprotection atposition 1 of the pyrrolidine ring, so long as the deprotection iscarried out under such conditions that the other functional groups andthe configuration are not changed. In the case where, for example, the1-position protective group is a benzyloxycarbonyl group, generally,catalytic reduction is performed by use of a palladium catalyst. In thiscase, ammonium formate or the like may be employed as a hydrogen sourcein place of hydrogen gas. Alternatively, the deprotection may beperformed through, for example, a method employing an organosilane(e.g., triethylsilane), a method employing a strong acid (e.g.,hydrobromic acid-acetic acid, trifluoroacetic acid, ortrifluoromethanesulfonic acid-trifluoroacetic acid), or a methodemploying sodium-liquid ammonia (Birch reduction conditions).Preferably, a palladium catalyst is employed in a hydrogen atmosphere.

In step 4, no particular limitation is imposed on the oxidation of thehydroxymethyl group, so long as the oxidation is performed undergenerally employed conditions. Examples of the oxidizing reagent to beemployed include Dess-Martin reagents, dimethyl sulfoxide (includingSwern oxidation), ruthenium reagents such as tetrapropylammoniumperruthenate (TPAP)/4-methylmorpholine N-oxide (NMO), and chromium oxidereagents such as pyridinium chlorochromate (PCC) and Collins reagent.Oxidation or post-reaction treatment is preferably performed under suchconditions that isomerization of the formyl group at position 4 of thepyrrolidine ring [i.e., isomerization from a (4S)-form to a (4R)-form]does not occur. When such isomerization occurs, an undesirebleenantiomer [i.e., (4R)-form] can be separated through chromatography ora similar technique. Among the aforementioned oxidation conditions,oxidation employing a Dess-Martin reagent is preferred. Preferably, theresultant product is employed in the subsequent step without beingpurified.

In step 5, the carbonyl group of the aldehyde moiety is subjected tomethylenation. The methylenation generally employs Wittig reaction.Preferably, the methylenation is performed under such conditions thatthe aforementioned isomerization does not occur. The methylenation maybe performed through a method employing an organotitanium reagent suchas Tebbe reagent, Grubbs reagent, or Nozaki-Rombert reagent. Preferably,diiodomethane is employed.

In step 6, the protective group at position 1 of the pyrrolidine ringmust be removed with retention of the carbon-carbon double bond of thevinyl or vinylidene group at position 4 of the pyrrolidine ring. Suchdeprotection is performed through, for example, a method employing astrong acid as described above in step 3, or a method employing bariumhydroxide. Preferably, the deprotection is performed through a methodemploying sodium-liquid ammonia.

In step 7, a carbene (or carbenoid) is added to the carbon-carbon doublebond to form a cyclopropane ring. Examples of the reagent to be employedfor this reaction include a diiodomethane/zinc-copper complex,diiodomethane/diethylzinc, diiodomethane/samarium iodide, adibromomethane/nickel complex, and a diazomethane/transition metalcomplex such as a diazomethane/palladium complex. Preferably, adiazomethane/palladium acetate complex is employed.

Step 8 can be performed under conditions similar to those of step 3.Step 8 is performed under such conditions that the cyclopropane ringdoes not undergo cleavage or rearrangement through catalytic reduction(catalytic hydrolysis) employing a transition metal catalyst.

In step 9, oxidation of the aldehyde moiety can be performed by use ofan oxidizing agent such as lead tetraacetate, a permanganate (e.g.,potassium permanganate), sodium periodate (with use of a rutheniumcatalyst), chromic acid, silver oxide, sodium hypochlorite-sodiumdihydrogenphosphate, or hydrogen peroxide. Of these oxidizing agents,sodium hypochlorite-sodium dihydrogenphosphate is preferred.

In step 10, esterification of the carboxylic moiety is performedthrough, for example, a method in which an alcohol is caused to act onthe carboxylic moiety in the presence of an acid catalyst such assulfuric acid, p-toluenesulfonic acid, or fluoroboric acid, anesterification method employing a dehydrating agent (a method in whichreaction proceeds via an active ester, or a method for activating analcohol), an O-alkylation method employing diazomethane or a similarreagent, a method employing an alkene or an alkyne in the presence of anacid catalyst, an alkylation method in which reaction proceeds via acarboxylic salt, or a method in which reaction proceeds via an acidanhydride or an acid halide. Of these methods, a method employingdiazomethane is preferred.

In step 11, an organometallic reagent is added to the ester, to therebyconvert the ester into a tertiary alcohol. Examples of the reagent to beemployed for this reaction include a Grignard reagent, an organolithiumreagent, an organocerium reagent, an organotitanium reagent, anorganozinc reagent, an organoaluminum reagent, an organocopper reagent,and an organosamarium reagent. The reaction is generally performed byuse of a solvent which is inert to organic metal (e.g., an ether solventsuch as diethyl ether or tetrahydrofuran). Of these organometallicreagents, a Grignard reagent, an organolithium reagent, or anorganocerium reagent is preferred, with a Grignard reagent being morepreferred. In order to prevent the aforementioned isomerization,preferably, the reaction is performed at a temperature of −30 to 0° C.

In step 12, the tertiary alcohol derivative is converted into an alkenethrough dehydration. The reaction may be generally employed dehydration(elimination) reaction. Examples of the reaction include dehydrationemploying an acid catalyst such as sulfuric acid or p-toluenesulfonicacid, dehydration employing alumina or the like as a catalyst,dehydration employing a sulfonating agent or an esterifying agent (e.g.,thionyl chloride-pyridine), and 1,2-elimination of an ester or asulfonic ester (e.g., causing a base to act on a substituted sulfonyloxygroup). Preferably, methanesulfonyl chloride is employed.

In step 13, the carbon-carbon double bond of the vinylidene group isconverted into an isopropyl group through catalytic reduction. Reductionof the carbon-carbon double bond may be performed simultaneously withdeprotection at position 1 of the pyrrolidine ring. In addition tocatalytic reduction employing a palladium catalyst, there may beperformed catalytic hydrogenation employing ammonium formate or the likein place of hydrogen gas. So long as the other functional groups are notaffected, there may be performed, for example, reduction employing anorganometallic salt/metal hydride reagent (e.g., cobalt chloride/sodiumhydride) or hydroboration-protonation. Preferably, a palladium-carboncatalyst is employed in a hydrogen atmosphere.

Step 14 is performed in a manner similar to that of step 3.

In step 15, the absolute configuration of the formyl group at position 4of the pyrrolidine ring is converted from a (4S)-form to a (4R)-form.This isomerization can be performed in an appropriate solvent by use ofan organic base (e.g., triethylamine or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)) or an inorganic base (e.g.,potassium carbonate or sodium hydroxide). Preferably, triethylamine isemployed. The reaction is preferably performed at a temperature of −78to 40° C. In the case where isomerization conversion is less than 100%,a necessary enantiomer [i.e., (4R)-form] can be separated and purifiedthrough chromatography or a similar technique.

Step 16 may be performed in a manner similar to that of step 5, butpreferably, methyltriphenylphosphonium bromide is employed. Step 17 isperformed in a manner similar to that of step 13, and step 18 isperformed in a manner similar to that of step 3. Steps 16 to 18 may beperformed simultaneously.

The compound (I) of the present invention can be produced from acompound represented by formula (VI-1) or (VI-2) and a compoundrepresented by formula (VII) through, for example, a method describedbelow. Now will be described the production method by taking, as anexample, production of Compound No. 1 described below in the Examplessection.

Compound (4) can be produced by dissolving compound (2) in anappropriate solvent, and then reacting compound (2) with3-(tert-butoxycarbonyl)amino-4-aliphatic-substituted-pyrrolidine (3) inthe presence of a base. Examples of the protective group which may beemployed include, in addition to a tert-butyloxycarbonyl (Boc) group, abenzyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group, an acetylgroup, a methoxyacetyl group, a trifluoroacetyl group, a pivaloyl group,a formyl group, a benzoyl group, a tert-butyl group, a benzyl group, atrimethylsilyl group, and an isopropyldimethylsilyl group. Examples ofthe base which may be employed include carbonates, hydrogencarbonates,and hydroxides of alkali metals and alkaline earth metals;trialkylamines such as triethylamine and N,N-diisopropylethylamine;pyridine; 1,8-diazabicycloundecene; and N-methylpiperidine. Preferably,triethylamine is employed. No particular limitation is imposed on thesolvent to be employed, so long as it does not impede reaction.N,N-dimethylformamide, dimethyl sulfoxide, sulfolane, acetonitrile,ethanol, dimethylacetamide, tetrahydrofuran, or N-methylpyrrolidone ispreferred, with dimethyl sulfoxide or sulfolane being particularlypreferred.

Subsequently, compound (4) is subjected to hydrolysis, followed bydeprotection of the amino protective group, to thereby yield thecompound (I-1) of the present invention. Hydrolysis of compound (4) canbe performed under generally employed conditions. For example, thehydrolysis can be performed by causing a base to act on compound (4) inan alcohol solvent (e.g., methanol or ethanol). The base is preferablytriethylamine. The reaction is preferably performed under ice cooling.The deprotection can be performed under conditions suitable for theemployed protective group. For example, the deprotection is performed bytreating the above-hydrolyzed product with concentrated hydrochloricacid. After completion of reaction, the reaction mixture is treatedwith, for example, an aqueous sodium hydroxide solution so that theresultant reaction mixture becomes basic.

The target product may be obtained by use of a carboxylic acid compoundin place of compound (4), which is a boron chelate.

The compound (I) of the present invention exhibits potent antibacterialactivity almost comparable to that of a known synthetic quinoloneantibacterial agent, and exhibits low convulsion-inducing effect andchromosomal-aberration-inducing effect and high safety. Therefore, thecompound can be employed as a drug for humans, animals, and fish or as apreservative for agricultural chemicals and food. When the compound ofthe present invention is employed as a drug for humans, the daily dosefor an adult is 50 mg to 1 g, preferably 100 to 500 mg. When thecompound is employed for veterinary purposes, the dose differs dependingon the purpose of the administration, the size of the animal to betreated, the type of the pathogenic bacteria infecting the animal, andthe severity of the infection. The daily dose is generally 1 to 200 mg,preferably 5 to 100 mg per kg (the weight of the animal). The daily doseis administered once a day, or 2 to 4 times a day in a divided manner.If necessary, the daily dose may exceed the aforementioned range.

The compound (I) of the present invention is active on a broad range ofmicroorganisms which cause various infectious diseases, and thus isuseful in treatment, prevention, or alleviation of pathologicalconditions caused by these pathogens. Examples of bacteria orbacteria-like microorganisms on which the compound of the presentinvention exhibits efficacy include those belonging to the genusStaphylococcus, Streptococcus pyogenes, hemolytic streptococci,Enterococcus, Streptococcus pneumoniae, the genus Peptostreptococcus,Neisseria gonorrhoeae, Escherichia coli, the genus Citrobacter, thegenus Shigella, Klebsiella pneumoniae, the genus Enterobacter, the genusSerratia, the genus Proteus, Pseudomonas aeruginosa, Haemophilusinfluenzae, the genus Acinetobacter, the genus Campylobacter, andChlamydia trachomatis.

Examples of pathological conditions caused by these pathogens includefolliculitis, furuncle, carbuncle, erysipelas, phelegmon, lymphangitisor lymphadenitis, panaritium, subcutaneous abscess, hidrosadenitis,aggregated acne, infectious atheroma, anal abscess, mastitis,superficial secondary infections caused by trauma, burn, operativewound, or similar wounds, laryngopharyngitis, acute bronchitis,tonsillitis, chronic bronchitis, bronchiectasis, diffusepanbronchiolitis, secondary infection caused by chronic respiratorydiseases, pneumonia, pyelonephritis, cystitis, prostatitis,epididymitis, gonococcal urethritis, non-gonococcal urethritis,cholecystitis, cholangitis, bacillary dysentery, enteritis, uterineadnexitis, intrauterine infection, bartholinitis, blepharitis,hordeolum, dacryocystitis, tarsadenitis, corneal ulcer, otitis media,sinusitis, periodontitis, pericoronitis, gnathitis, peritonitis,endocarditis, sepsis, meningitis, and skin infectious diseases.

Examples of acid-fast bacteria on which the compound (I) of the presentinvention exhibits efficacy include members of the so-calledMycobacterium tuberculosis complex (Mycobacterium tuberculosis, M.bovis, M. africanum) and atypical acid-fast bacteria (M. kansasii, M.marinum, M. scrofulaceum, M. avium, M. intracellulare, M. xenopi, M.fortuitum, M. chelonae). Acid-fast bacterial infectious diseases causedby any of these pathogens are broadly categorized into three groups;i.e., tuberculosis, atypical acid-fast bacterial disease, and lepra,based on the identity of the causal bacterium. Mycobacteriumtuberculosis infections can be seen not only in the lungs, but also inthe thoracic cavity, trachea/bronchi, lymph nodes, systemicallydisseminated, bone joints, meninges or brain, digestive organs(intestine or liver), skin, mammary gland, eye, middle ear or throat,urinary tract, male genital organs, and female genital organs. Atypicalacid-fast bacteriosis (nontuberculous mycobacteriosis) is primarilyfound in the lung, but also found in local lymphadenitis, skin softtissue, bone joints, and systemic disseminated pathological condition.

The compound of the present invention is effective on a variety ofmicroorganisms which cause infectious disease in animals. Examples ofsuch microorganisms include those belonging to the genus Escherichia,the genus Salmonella, the genus Pasteurella, the genus Haemophilus, thegenus Bordetella, the genus Staphylococcus, and the genus Mycoplasma.Specific examples of diseases include, in birds, Escherichia coliinfections, pullorum disease, avian paratyphoid, fowl cholera,infectious coryza, staphylococcosis, and mycoplasma infections; in pigs,Escherichia coli infections, salmonellosis, pasteurellosis, Haemophilusinfections, atrophic rhinitis, exudative epidermitis, mycoplasmainfections; in cattle, Escherichia coli infections, salmonellosis,hemorrhagic septicemia, mycoplasma infections, contagious bovinepleuropneumonia, and mastitis; in dogs, Escherichia coli septicemia,salmonella infections, hemorrhagic septicemia, uterine empyema, andcystitis; and in cats, exudative pleurisy, cystitis, chronic rhinitis,haemophilus infections, kitten diarrhea, and mycoplasma infections.

Antibacterial drugs containing the compound (I) of the present inventioncan be prepared by selecting a suitable drug form in consideration ofthe manner of administration and using any of ordinarily employedpreparation methods. Examples of the form of the antibacterial drugscontaining the compound of the present invention include tablets,powders, granules, capsules, solutions, syrups, elixirs, oil or aqueoussuspensions. Injection drugs may contain a stabilizer, a preservative,or a solubilizing agent. Alternatively, a solution which may contain anyof these additives may be placed in a container and converted into solidthrough, for example, freeze-drying, and the thus-prepared solidpreparation may be restituted before use. In this connection, a singledose or a plurality of doses may be contained into one container.Exemplary external application forms include solutions, suspensions,emulsions, ointments, gels, creams, lotions, and sprays. Solidpreparations may contain pharmaceutically acceptable additives alongwith the active compound. Examples of such additives include fillers,binders, disintegrators, dissolution accelerators, humectants, andlubricants. Exemplary liquid preparation forms include solutions,suspensions, and emulsions, and they may contain as an additive asuspending agent, an emulsifier, or the like.

Next will be described formulation examples of antibacterial drugscontaining the compound of the present invention.

FORMULATION EXAMPLE 1 Capsule

Compound of Example 1 100.0 mg Cornstarch 23.0 mg CMC calcium 22.5 mgHydroxymethyl cellulose 3.0 mg Magnesium stearate 1.5 mg Total 150.0 mg

FORMULATION EXAMPLE 2 Solution

Compound of Example 2 1 to 10 g Acetic acid or sodium hydroxide 0.5 to 2g Ethyl p-hydroxybenzoate 0.1 g Purified water 87.9 to 98.4 g Total 100g

FORMULATION EXAMPLE 3 Powder for Mixing with Feed

Compound of Example 3 1 to 10 g Cornstarch 89.5 to 98.5 g Lightanhydrous silicic acid 0.5 g Total 100 g

EXAMPLE

The present invention will next be described in more detail by way ofReferential Examples and Examples, which should not be construed aslimiting the invention thereto.

Referential Example 1(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(p-toluenesulfonyloxy)methylpyrrolidine

(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-hydroxymethylpyrrolidine(5.00 g, 14.3 mmol) was dissolved in pyridine (50 mL). p-Toluenesulfonylchloride (4.08 g, 21.4 mmol) and 4-(N,N-dimethyl)aminopyridine (174 mg,1.43 mmol) were added thereto at room temperature, and the resultantmixture was stirred under a nitrogen atmosphere for 24 hours. Undercooling, ethyl acetate (200 mL) and 1 mol/L hydrochloric acid (200 mL)were added thereto, followed by extraction with ethyl acetate (200mL×2). The organic layer was washed sequentially by 1 mol/L hydrochloricacid (100 mL), saturated sodium hydrogencarbonate (100 mL), andsaturated brine (100 mL), and dried over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure. Theresidue was purified through silica gel column chromatography (ethylacetate:n-hexane=1:1), to thereby yield the title compound as acolorless amorphous substance (6.27 g, 87%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.42(9H,s),2.44(3H,s),2.58-2.68(1H,m),3.17-3.36(2H,m),3.59-3.69(2H,m),3.90-4.08(1H,m),4.22(1H,dd,J=4.9,10.5Hz),4.25-4.38(1H,m),4.60(1H,d,J=29.1Hz),5.11(2H,s),7.27-7.42(7H,m),7.76(2H,d,J=8.1 Hz).

MS(EI)m/z:405(M-Boc)⁺.

Referential Example 2(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine

Under cooling with ice, a solution of methyllithium (121 mL, 1.02 M,diethyl ether solution) was added to a suspension of copper(I) iodide(11.8 g, 62.1 mmol) in diethyl ether (200 mL), followed by stirring for30 minutes. The temperature of the reaction mixture was cooled to −78°C. A solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(p-toluenesulfonyloxy)methylpyrrolidine(6.27 g, 12.4 mmol) in diethyl ether (140 mL) was added thereto. Thetemperature of the mixture was elevated gradually, followed by stirringfor 1 hour under cooling with ice. Under cooling with ice, a saturatedaqueous ammonium chloride solution (100 mL) and 28% aqueous ammonia (25mL) were added to the resultant mixture, and then water (100 mL) wasadded thereto, followed by extraction with ethyl acetate (300 mL×2). Thecombined organic layer was washed by saturated brine (100 mL), and driedover anhydrous sodium sulfate. The filtrate was concentrated underreduced pressure. The residue was purified through silica gel columnchromatography (ethyl acetate:n-hexane=1:3 to 1:2), to thereby yield thetitle compound as a colorless oily substance (3.09 g, 71%).

¹H-NMR(400 MHz,CDCl₃)δppm:0.93-0.97(3H,m),1.44(9H,s),1.27-1.55(2H,m),2.09-2.23(1H,m),3.02(1H,q,J=11.1Hz),3.41-3.68(3H,m),4.24(1H,brs),4.47-4.64(1H,m),5.07-5.18(2H,m),7.28-7.42(5H,m).

IR(ATR)ν cm⁻¹: 3321,2966,2937,2877,1684,1525,1454,1417,1363,1331,1244,1161.

[α]_(D) ^(19.5) −2.49° (c1.000,CHCl₃).

MS(FAB+)m/z:349(M+1)⁺.

HRMS (FAB+) m/z: Calcd for C₁₉H₂₈N₂O₄+H: 349.2127. Found: 349.2117.

Referential Example 3(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-ethylpyrrolidine

A 10% palladium carbon catalyst (50 mg) was added to a solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine(514 mg, 1.48 mmol) in methanol (15 mL). The resultant mixture wasstirred at room temperature for 2 hours under an ordinary pressure in ahydrogen atmosphere. After filtration, the filtrate was concentratedunder reduced pressure, to thereby yield the title compound as acolorless oily substance (324 mg, quantitative amount).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.94(3H,t,J=7.4Hz),1.21-1.33(1H,m),1.44(9H,s),1.40-1.54(1H,m),1.95-2.08(1H,m),2.53(1H,t,J=10.0Hz),2.79(1H,dd,J=3.1,11.1 Hz),3.12-3.24(2H,m),4.16(1H,brs),4.75(1H,brs).

MS(EI)m/z:215(M+1)⁺.

Example 17-[(3S,4S)-3-Amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 1)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (484 mg, 1.34 mmol) and triethylamine (224μL, 1.61 mmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine (324 mg, 1.48mmol) in dimethyl sulfoxide (5 mL), and the mixture was stirred at roomtemperature for 4 hours. Subsequently, water (100 mL) was added to thereaction mixture, and a precipitated yellow solid was collected throughfiltration. Ethanol (10 mL) and water (2.5 mL) were added so as todissolve the solid, and triethylamine (2.5 mL) was added to thesolution, followed by refluxing in an oil bath at 90° C. for 4 hours.The reaction mixture was concentrated under reduced pressure, and a 10%aqueous citric acid solution (20 mL) was added thereto, followed byextraction with chloroform (50 mL×2). The thus-obtained organic layerwas dried over anhydrous sodium sulfate. After filtration, the filtratewas concentrated under reduced pressure to thereby obtain a yellowsolid. Subsequently, concentrated hydrochloric acid (10 mL) was added tothe solid at room temperature, followed by stirring for 30 minutes. Thereaction mixture was washed by chloroform (50 mL×3), and the pH of theobtained aqueous layer was adjusted to 11.0 with a 10 mol/L aqueoussodium hydroxide solution under cooling with ice. The pH was adjustedagain to 7.4. The resultant mixture was extracted sequentially with amethanol-chloroform mixture (1:4) (200 mL×2), and chloroform (100 mL).The combined organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue wasrecrystallized from an ethanol-28% aqueous ammonia mixture (6:1),followed by drying under reduced pressure, to thereby yield the titlecompound as yellow crystals (441 mg, 80%).

mp:217-219° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δppm:0.93-1.02(3H,m),1.36-1.57(4H,m),2.07-2.21(1H,m),3.34(1H,d,J=10.0Hz),3.48-3.62(6H,m),3.83(1H,s),3.95-4.04(1H,m),5.01(1H,dm,J=63.5Hz),7.64(1H,d,J=14.6 Hz),8.40(1H,d, J=2.2 Hz).

IR(ATR)ν cm⁻¹:3388,3078,3037,2952,2873,1724,1618,1510,1435,1367,1327,1271,1230.

[α]_(D) ^(24.2) −121.03° (c0.397,0.1 mol/L NaOH).

MS(EI)m/z:408(M+1)⁺.

Anal.:Calcd for C₂₀H₂₃F₂N₃O₄.0.25H₂O: C, 58.32; H, 5.75; N, 10.20%.Found: C, 58.49; H, 5.67%; N, 10.29.

Example 27-[(3S,4S)-3-Amino-4-ethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 2)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (455 mg, 1.32 mmol) and triethylamine (221μL, 1.58 mmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine (311 mg, 1.45mmol) in sulfolane (4 mL), and the mixture was stirred in an oil bath at45° C. for 48 hours. Subsequently, ethanol (20 mL), water (5 mL), andtriethylamine (5 mL) were added to the reaction mixture, followed byrefluxing in an oil bath at 90° C. for 5 hours. The reaction mixture wasconcentrated under reduced pressure, and a 10% aqueous citric acidsolution (30 mL) was added thereto, followed by extraction with ethylacetate (100 mL×2). The thus-obtained organic layer was washedsequentially by water (50 mL×3) and saturated brine (50 mL), and driedover anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. Subsequently, concentratedhydrochloric acid (10 mL) was added to the obtained bark oily substanceat room temperature, and the reaction mixture was washed by chloroform(50 mL×3), and the pH of the obtained aqueous layer was adjusted to 12.0with a 10 mol/L aqueous sodium hydroxide solution under cooling withice. The pH was adjusted again to 7.4. The resultant mixture wasextracted with chloroform (200 mL×2). The combined organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The obtained residue was recrystallized from ethanol,and dried under reduced pressure, to thereby yield the title compound aspale bark crystals (65.0 mg, 12%).

mp:214-216° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.97(3H,t,J=7.4Hz),1.15-1.28(1H,m),1.41-1.66(3H,m),2.14-2.25(1H,m),2.42(3H,s),3.13(1H,d,J=10.3Hz),3.29(1H,t,J=8.7 Hz),3.49-3.60(2H,m),3.99-4.09(2H,m),5.02(1H,dm,J=64.5 Hz), 7.65(1H,d,J=14.4Hz),8.43(1H,d,J=3.2 Hz).

IR(ATR)ν cm⁻¹:3390,3084,3037,2964,2912,1712,1616,1510,1468,1435,1350,1308.

MS(EI)m/z:392(M+1)⁺.

Anal.:Calcd for C₂₀H₂₃F₂N₃O₃.0.25H₂O: C, 60.67; H, 5.98; N, 10.61; F,9.60. Found: C, 60.85; H, 5.89; N, 10.53; F, 9.55.

Example 3(3S)-10-[(3S,4S)-3-Amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Compound No. 3)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (193 mg, 587 μmol) and triethylamine (98.2μL, 704 μmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine (138 mg, 644μmol) in dimethyl sulfoxide (3 mL), and the mixture was stirred at roomtemperature for 16 hours. The reaction mixture was concentrated underreduced pressure, and ethanol (4 mL) and water (1 mL) were added so asto dissolve the concentrated product. Subsequently, triethylamine (1 mL)was added to the solution, followed by refluxing in an oil bath at 90°C. for 2 hours. The reaction mixture was concentrated under reducedpressure, and dissolved in chloroform (50 mL). The solution was washedby a 10% aqueous citric acid solution (30 mL). The obtained organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Subsequently, concentratedhydrochloric acid (6 mL) was added to the obtained yellow solid at roomtemperature, followed by stirring for 30 minutes. The reaction mixturewas transferred to a separatory funnel with 1 mol/L hydrochloric acid(20 mL), followed by washing with chloroform (50 mL). Subsequently, thepH of the obtained aqueous layer was adjusted to 12.0 with a 10 mol/Laqueous sodium hydroxide solution under cooling with ice. The pH wasadjusted again to 7.4. The resultant mixture was extracted withchloroform (200 mL×2). The combined organic layer was dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure. The obtained residue was recrystallized from an ethanol-28%aqueous ammonia (10:1) solvent mixture, and dried under reducedpressure, to thereby yield the title compound as yellow crystals (176mg, 79%).

mp:239-241° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.95(3H,t,J=7.3 Hz),1.49(3H,d,J=6.6Hz),1.34-1.51(2H,m),2.06(1H,brs),3.31(1H,d,J=10.5Hz),3.43-3.60(3H,m),3.90-4.00(1H,m),4.24(1H,d,J=11.2Hz),4.42(1H,d,J=11.5 Hz),4.50-4.60(1H,m),7.45(1H,d,J=14.2Hz),8.30(1H,s).

IR(ATR)ν cm⁻¹:3388,3035,2972,2871,1697,1612,1520,1431,1400,1344,1315,1273,1203.

[α]_(D) ^(24.8) −207.15° (c0.390,0.1 mol/L NaOH).

MS(EI)m/z:376(M+1)⁺.

Anal.:Calcd for C₁₉H₂₂FN₃O₄.0.25H₂O: C, 60.07; H, 5.97; N, 11.06; F,5.00. Found: C, 60.30; H, 5.86; N, 11.12; F, 5.02.

Referential Example 4(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-propylpyrrolidine

Under a nitrogen atmosphere, ethylmagnesium bromide (3.6 mL, 3.2 mmol,0.89M tetrahydrofuran solution) was added dropwise to a suspension ofcopper iodide (38 mg) in diethyl ether (1 mL) at −30° C. Subsequently, asolution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(p-toluenesulfonyloxy)methylpyrrolidine(400 mg, 0.792 mmol) in diethyl ether (4 mL) was added dropwise thereto.The mixture was stirred for 2 hours while the temperature thereof wasgradually elevated to 10° C. A saturated aqueous ammonium chloridesolution and 28% aqueous ammonia were added sequentially thereto, andthen the mixture was stirred until insoluble matter was dissolved. Theresultant mixture was extracted with diethyl ether (100 mL×2), washed bysaturated brine (100 mL), and dried over anhydrous magnesium sulfate.After filtration, the filtrate was concentrated through solvent removal.The residue was purified through silica gel column chromatography(n-hexane:ethyl acetate=4:1), to thereby yield the title compound ascolorless crystals (106 mg, 36.9%).

¹H-NMR(400 MHz,CDCl₃)δppm:0.90-0.92(3H,m),1.24-1.45(13H,m),2.20-2.30(1H,m),3.00(1H,q,J=10.5Hz),3.40-3,68(3H,m),4.18-4.25(1H,m),4.55(1H,br),5.12(2H,s),7.31-7.38(5H,m).

IR(ATR)ν cm⁻¹:3282,2958,2873,1698,1681, 1666,1413,1162,1060,696.

HRMS(FAB)m/z:Calcd for C₂₀H₃₁O₄N₂: 363.2284. Found: 363.2266.

Referential Example 5(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-propylpyrrolidine

A mixture of (3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-propylpyrrolidine(4.02 g, 11.09 mmol) and a 10% palladium carbon catalyst (1.0 g) inethanol (100 mL) was stirred hard for 20 hours under an ordinarypressure in a hydrogen atmosphere. Insoluble matter was removed throughfiltration by use of Celite, and then ethanol was removed under reducedpressure. The residue was dissolved in diethyl ether (200 mL), and thesolution was washed by a 1 mol/L aqueous sodium hydroxide solution (100mL), followed by drying over anhydrous sodium sulfate. After filtration,the filtrate was concentrated under reduced pressure, to thereby yieldthe title compound as a colorless oily substance (2.34 g, 92.4%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.91(3H,t,J=7.0Hz),1.18-1.45(13H,m),1.76(1H,br),2.06-2.13(1H,m),2.50(1H,t,J=10.0Hz),2.77(1H,dd,J=3.0,11.0Hz),3.12-3.22(2H,m),4.10-4.16(1H,m),4.65(1H,br).

MS(ESI)m/z:229(M⁺+1).

Example 47-[(3S,4S)-3-Amino-4-propylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 4)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (505 mg, 1.40 mmol) and triethylamine (1.0mL) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-propylpyrrolidine (342 mg, 1.50mmol) in dimethyl sulfoxide (3 mL), followed by stirring at roomtemperature for 7 days. Subsequently, water (100 mL) was added to thereaction mixture, and a precipitated yellow solid was collected throughfiltration. Ethanol (45 mL) and water (5 mL) were added so as todissolve the solids, and triethylamine (10 mL) was added to thesolution, followed by refluxing for 2 hours. The reaction mixture wasconcentrated under reduced pressure, and a 10% aqueous citric acidsolution (20 mL) was added thereto, followed by extraction withchloroform (50 mL×2). The combined organic layer was washed by saturatedbrine (50 mL), and the thus-obtained organic layer was dried overanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. Subsequently, concentratedhydrochloric acid was added so as to dissolve the obtained residue atroom temperature, followed by stirring for 30 minutes. The reactionmixture was washed by chloroform (50 mL×3), and the pH of the obtainedaqueous layer was adjusted to 11.0 with a 10 mol/L aqueous sodiumhydroxide solution under cooling with ice. The pH was adjusted again to7.5. The resultant mixture was extracted with chloroform (200 mL×3). Thecombined organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The obtained residuewas recrystallized from a mixture of 2-propanol and a small amount ofethanol, and dried under reduced pressure, to thereby yield the titlecompound as orange-yellow crystals (380 mg, 63.7%).

mp:79-83° C.

¹H-NMR(400 MHz,DMSO-d₆)δ ppm:0.92(3H,t,J=7.0Hz),1.27-1.54(6H,m),2.05-2.11(1H,m),3.22-3.39(1H,m),3.43-3.48(2H,m),3.52(3H,s),3.61(1H,dt,J=4.0,10.0Hz),3.90(1H,dt,J=4.5, 10.5 Hz),4.05(1H,dt,J=5.5,9.0Hz),5.00-5.20(1H,m),7.63(1H,d,J=14.0 Hz),8.58(1H,d,J=3.5 Hz).

IR(ATR)ν cm⁻¹:3383,2929,2871,1616,1541,1429,1313,1051,928,804.

[α]_(D) −226.08° (c0.265,DMSO).

Anal.:Calcd for C₂₁H₂₅F₂N₃O₄.0.25H₂O: C, 59.22; H, 6.03; N, 9.87; F,8.92. Found: C, 59.18; H, 6.01; N, 9.92; F, 9.11.

Example 57-[(3S,4S)-3-Amino-4-propylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid.hydrochloride (Compound No. 5)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (455 mg, 1.32 mmol) and triethylamine (1.5mL) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-propylpyrrolidine (731 mg, 3.20mmol) in dimethyl sulfoxide (5 mL), and the mixture was stirred at roomtemperature under a nitrogen atmosphere for 2 weeks. The reactionmixture was concentrated under reduced pressure. Subsequently, water wasadded to the residue, and precipitated crystals were collected throughfiltration. Ethanol (45 mL), water (5 mL), and triethylamine (10 mL)were added to the obtained crystals, followed by refluxing for 3 hours.The reaction mixture was concentrated under reduced pressure, and theresidue was dissolved in chloroform (100 mL). The obtained organic layerwas washed sequentially by a 10% aqueous citric acid solution (50 mL)and saturated brine (50 mL), dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. Subsequently,concentrated hydrochloric acid was added at room temperature so as todissolve the obtained residue. The reaction mixture was washed bychloroform (50 mL×3), and the pH of the obtained aqueous layer wasadjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solution undercooling with ice. The pH was adjusted again to 7.6. The resultantmixture was extracted with chloroform (200 mL×2). The combined organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The obtained residue was purifiedthrough fractional thin-layer chromatography(chloroform:methanol:water=7:3:1, developer). The obtained crystals weredissolved in concentrated hydrochloric acid, and the solution wasconcentrated under reduced pressure. The obtained residue wasrecrystallized from 2-propanol, to thereby yield the title compound asan orange crystals (190 mg, 13.2%).

mp:197-207° C.

¹H-NMR(400 MHz,D₂O)δ ppm:0.90(3H,t,J=6.5Hz),1.08-1.60(6H,m),2.13-2.21(1H,m),2.35(3H,s),3.04(1H,d,J=9.5Hz),3.17(1H,t,J=8.5 Hz),3.39-3.42(1H,m),3.50(1H,t,J=9.0Hz),3.88-4.02(2H,m),4.90-5.07(1H,m),7.61(1H,d,J=14.0 Hz),8.41(1H,d,J=3.0Hz).

IR(ATR)ν cm⁻¹:2870,1716,1614,1427,1315,1128,1026,926,806.

[α]_(D) −299.45° (c0.309,0.1 mol/L NaOH).

Anal.:Calcd for C₂₁H₂₅F₂N₃O₃.HCl.0.5H₂O.0.5C₃H₇OH: C, 56.19; H, 6.50; N,8.74; F, 7.90; Cl, 7.37. Found: C, 55.88; H, 6.45; N, 8.90; F, 8.11; Cl,7.60.

Example 6(3S)-10-[(3S,4S)-3-Amino-4-propylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid.hydrochloride (Compound No. 6)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (1.65 g, 5.00 mol) and triethylamine (2 mL)were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-propylpyrrolidine (1.26 g, 5.52mmol) in dimethyl sulfoxide (10 mL), followed by stirring at roomtemperature for 6 days. The reaction mixture was concentrated underreduced pressure. Subsequently, water was added thereto, andprecipitated crystals were collected through filtration. Ethanol (90 mL)and water (10 mL) were added so as to dissolve the obtained crystals,and triethylamine (20 mL) was added to the solution, followed byrefluxing for 3 hours. The reaction mixture was concentrated underreduced pressure, and dissolved in chloroform (300 mL). The solution waswashed sequentially by a 10% aqueous citric acid solution (200 mL) andsaturated brine (100 mL). The obtained organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. Subsequently, concentrated hydrochloric acid was added so asto dissolve the obtained yellow solid at room temperature, and thesolution was stirred for 30 minutes. The reaction mixture was washed bychloroform (50 mL), and the thus-obtained aqueous layer was diluted.Subsequently, the precipitated crystals were collected throughfiltration, recrystallized from ethanol, and dried over under reducedpressure, to thereby yield the title compound as yellow crystals (1.44g, 59.4%). Separately, the pH of the above-obtained aqueous layeracidified with hydrochloric acid was adjusted to 7.7 with an aqueoussodium hydroxide solution. The resultant mixture was extracted withchloroform (100 mL×2), and dried over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure. Theobtained crystals were recrystallized from 2-propanol, to thereby yieldthe title compound as a free form (90 mg, 4.6%).

Hydrochloride (crystallized with ethanol):

mp:225-231° C.

¹H-NMR(400 MHz,DMSO-d₆)δ ppm:0.91(3H,t,J=7.0Hz),1.26-1.49(7H,m),1.98-2.04(1H,m),3.30-3.40(2H,m),3.52-3.58(1H,m),3.73(1H,dt,J=4.0,10.0Hz),4.01-4.07(1H,m),4.21(1H,dd,J=1.5,11.0 Hz),4.49(1H,dd,J=1.5,11.5 Hz),4.81-4.87(1H,m),7.49(1H,d,J=14.5 Hz),8.84(1H,s).

IR(ATR)ν cm⁻¹:3388,3035,2954,2871,1705,1614,1433,1354, 1115,980,804.

Anal.:Calcd for C₂₀H₂₄FN₃O₄.HCl.0.75H₂O: C, 54.67; H, 6.08; N, 9.56; F,4.32; Cl, 8.07. Found: C, 54.35; H, 5.76; N, 9.56; F, 4.40; Cl, 7.68.

Free form (crystallized with 2-propanol):

mp:213-217° C.

[α]_(D)−257.88° (c0.264,DMSO).

Anal.:Calcd for C₂₀H₂₄FN₃O₄: C, 61.68; H, 6.21; N, 10.79; F, 4.88.Found: C, 61.35; H, 6.02; N, 10.66; F, 4.90.

Referential Example 6(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-formylpyrrolidine

(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-hydroxymethylpyrrolidine(1.00 g, 2.85 mmol) was dissolved in dichloromethane (40 mL). Undercooling with ice, Dess Martin reagent (1.42 g, 3.42 mmol) was addedthereto, and the resultant mixture was stirred at room temperature for 2hours under a nitrogen atmosphere. Under cooling, 5% aqueous sodiumthiosulfate solution (50 mL) was added thereto, followed by stirring for0.5 hours. The aqueous layer was extracted with dichloromethane (30mL×1), and the organic layer was washed sequentially by a 0.05 mol/Laqueous sodium hydroxide solution (60 mL) and saturated brine (100 mL),and dried over anhydrous sodium sulfate. After filtration, the filtratewas concentrated under reduced pressure, to thereby yield the titlecompound as a colorless oily substance (0.90 g, 91%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.42(9H,s),3.20-3.52(2H,m),3.53-3.65(1H,m),3.65-3.77(1H,m),3.77-3.95(1H,m),4.64(1H,s),4.90-5.21(3H,m),7.24-7.50(5H,m),9.77(1H,s).

MS(ESI)m/z:249(M-Boc)⁺.

Referential Example 7(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine

Under cooling with ice, titanium tetrachloride (3.30 mL, 1.0M toluenesolution) was added to a suspension of zinc powder (1.86 g, 28.5 mmol)in tetrahydrofuran (15 mL) over 2 minutes, followed by stirring for 5minutes at the same temperature. A solution of diiodomethane (1.15 mL,14.3 mL) in tetrahydrofuran (5 mL) was added thereto over 8 minutes, andthe resultant mixture was stirred for 15 minutes while the temperaturethereof was elevated to room temperature. A solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-formylpyrrolidine(900 mg, 2.58 mmol) in tetrahydrofuran (5.0 mL) was added thereto over 7minutes, followed by stirring for 1.5 hours. Subsequently, under coolingwith ice, the resultant mixture was poured to 0.5 mol/L hydrochloricacid (10 mL), followed by dilution with diethyl ether (60 mL). Theobtained aqueous layer was extracted with diethyl ether (100 mL×2). Thecombined organic layer was washed by saturated brine (100 mL), driedover anhydrous sodium sulfate, filtered, concentrated under reducedpressure. The obtained residue was purified through silica gel columnchromatography (ethyl acetate:n-hexane=1:3), to thereby yield the titlecompound as a colorless oily substance (3.09 g, 31%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.43(9H,s),2.89-3.02(1H,m),3.25-3.48(2H,m),3.56-3.74(2H,m),4.25(1H,s),4.57(1H,s),5.06-5.30(4H,m),5.71-5.84(1H,m),7.28-7.39(5H,m).

MS(ESI+)m/z:347(M+1)⁺.

Referential Example 8(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-vinylpyrrolidine

A solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine(275 mg, 0.794 mmol) in tetrahydrofuran (5 mL) and sodium (300 mg, 13.0mmol) were sequentially added to liquid ammonia (80 mL) at −78° C., andthe mixture was stirred at the same temperature for 0.5 hours. Thesolvent was removed, followed by dilution with chloroform (80 mL). Theresultant mixture was washed sequentially by 1 mol/L sodium hydroxide(30 mL) and saturated brine (100 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure, to therebyyield the title compound as a colorless oily substance (168 mg, 100).

¹H-NMR(400 MHz,CDCl₃)δppm:1.44(9H,s),2.70-2.89(3H,m),3.13-3.33(2H,m),4.13(1H,s),4.60(1H,s),5.09-5.21(2H,m),5.80(1H,ddd,J=7.4,0.5,17.6Hz).

MS(ESI)m/z:213(M+1)⁺.

Example 7(3S)-10-[(3S,4S)-3-Amino-4-vinyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Compound No. 7)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (233 mg, 709 μmol) and triethylamine (120 μL,851 μmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine (180 mg, 780μmol) in dimethyl sulfoxide (3.5 mL), followed by stirring at roomtemperature for 16 hours. Subsequently, water (10 mL) was added to thereaction mixture, and precipitated crystals were collected throughfiltration. The thus-collected crystals were suspended in a mixture ofethanol (10 mL) and water (2 mL). Thereafter, triethylamine (3 mL) wasadded thereto, followed by refluxing in an oil bath at 90° C. for 5hours. The reaction mixture was concentrated under reduced pressure, anddissolved in chloroform (100 mL). The resultant solution was washed by a10% aqueous citric acid solution (30 mL), and the obtained organic layerwas dried over anhydrous sodium sulfate, filtered, concentrated underreduced pressure. Subsequently, concentrated hydrochloric acid (5 mL)was added to the obtained yellow solid under cooling with ice, followedby stirring for 45 minutes. The reaction mixture was transferred to aseparatory funnel with 4 mol/L hydrochloric acid (5 mL), followed bywashing with chloroform (50 mL×7). The pH of the obtained aqueous layerwas adjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solutionunder cooling with ice. The pH was adjusted again to 7.4. The resultantmixture was extracted with chloroform (70 mL×3), and the combinedorganic layer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The obtained residue wasrecrystallized from ethanol, and dried under reduced pressure, tothereby yield the title compound as yellow crystals (188 mg, 64%).

mp:238-240° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:1.52(3H,d,J=6.9Hz),2.91-3.00(1H,m),3.41(1H,dq,J=10.4,2.2 Hz),3.56(1H,dd,J=10.0,5.6 Hz),3.71-3.76(2H,m),3.92(1H,ddd,J=10.5,5.9,2.4 Hz), 4.30(1H,dd,J=11.4,2.3Hz),4.48(1H,dd,J=11.4,2.1 Hz),4.59(1H,d,J=7.1Hz),5.23-5.32(2H,m),5.98(1H,ddd,J=17.8,10.1,7.3 Hz), 7.52(1H,d,J=14.2Hz),8.32(1H,s).

IR(ATR)ν cm⁻¹:3374,2983,2914,1709,1618,1525,1464,1396,1352,1309,1275,1211.

[α]_(D) ^(24.8) −115.0° (c0.100,0.1 mol/L NaOH).

MS(ESI)m/z:374(M+1)⁺.

Anal.:Calcd for C₁₉H₂₂FN₃O₄: C, 61.12; H, 5.40; N, 11.25; F, 5.09.Found: C, 61.04; H, 5.32; N, 11.14; F, 4.95.

Referential Example 9(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-cyclopropylpyrrolidine

An excessive amount of a solution of diazomethane in diethyl ether (30mL) was added to(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine(270 mg, 779 μmol). Under cooling with ice, palladium acetate (10 mg)was added thereto, followed by stirring at room temperature for 2 hours.After completion of reaction, insoluble matter was removed throughfiltration. The filtrate was concentrated under reduced pressure, andthe obtained residue was purified though silica gel columnchromatography (ethyl acetate:n-hexane=1:9 to 1:4), to thereby yield thetitle compound as a colorless oily substance (269 mg, 96%).

¹H-NMR(400 MHz,CDCl₃)δppm:0.08-0.29(2H,m),0.46-0.71(3H,m),1.45(9H,s)1.40-1.60(1H,m),3.27-3.41(2H,m),3.52-3.72(2H,m),4.24(1H,brs),4.70-4.90(1H,m),5.10-5.17(2H,m),7.28-7.40(5H,m).

MS(EI)m/z:388(M+Na)⁺.

Referential Example 10(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-cyclopropylpyrrolidine

A 10% palladium carbon catalyst (50 mg) was added to a solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-cyclopropylpyrrolidine(408 mg, 1.13 mmol) in ethanol (5 mL), followed by stirring in ahydrogen atmosphere under an ordinary pressure at 45° C. for 12 hours.After filtration, the filtrate was concentrated under reduced pressure,to thereby yield the title compound as a colorless oily substance (255mg, quantitative amount). The product was employed, without furtherpurification, in the subsequent step.

MS(EI)m/z:227(M+1)⁺.

Example 87-[(3S,4S)-3-Amino-4-cyclopropylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 8)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (180 mg, 499 μmol) and triethylamine (83 μL,598 μmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-cyclopropylpyrrolidine (113 mg,499 μmol) in acetonitrile (1 mL), and the mixture was stirred at roomtemperature for 15 hours. The reaction mixture was concentrated underreduced pressure. Ethanol (5 mL) and water (1 mL) were added so as todissolve the concentrated product. Subsequently, triethylamine (1 mL)was added to the solution, followed by refluxing in an oil bath at 100°C. for 4 hours. The reaction mixture was concentrated under reducedpressure, and concentrated hydrochloric acid (2 mL) was added thereto atroom temperature, followed by stirring for 15 minutes. Subsequently,water (10 mL) was added to the reaction mixture, followed by washingwith chloroform (20 mL). The pH of the obtained aqueous layer wasadjusted to 12.0, and the resultant mixture was washed by chloroform (5mL). The pH of the obtained aqueous layer was adjusted to 7.4, followedby extraction with chloroform (50 mL×3). The obtained organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The obtained residue was recrystallized from anethanol-28% aqueous ammonia mixture (8:1), and dried under reducedpressure, to thereby yield the title compound as pale yellow crystals(125 mg, 60%).

mp:211-214° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δppm:0.09-0.18(1H,m),0.30-0.38(1H,m),0.43-0.52(1H,m),0.55-0.64(1H,m),0.73-0.83(1H,m),1.20(1H,t,J=7.2Hz),1.32(1H,J=8.1Hz),1.54-1.72(2H,m),3.33-3.43(1H,m),3.52(1H,m),3.59(3H,s),3.67(2H,d,J=8.6 Hz),3.76(1H,t,J=8.3Hz),4.04-4.12(1H,m),4.84-5.06(1H,m),7.68(1H,d,J=14.7 Hz),8.49(1H,s).

IR(ATR)ν cm⁻¹:2954,2885,1988,1610,1568,1523,1495,1454,1377,1356,1288,1267.

[α]_(D) ^(24.6) +126.60° (c0.407,0.1 mol/L NaOH).

MS(EI)m/z:420(M+1)⁺.

Anal.:Calcd for C₂₁H₂₃F₂N₃O₄.0.5H₂O: C, 58.87; H, 5.65; N, 9.81. Found:C, 58.8; H, 5.78; N, 9.42.

Example 9(3S)-10-[(3S,4S)-3-Amino-4-cyclopropylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Compound No. 9)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid difluoroboran complex (244 mg, 741 μmol)and triethylamine (206 μL, 1.48 mmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-cyclopropylpyrrolidine (167 mg,741 μmol) in dimethyl sulfoxide (3 mL), and the resultant mixture wasstirred at room temperature for 16 hours.(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (244 mg, 741 μmol) was further added thereto,followed by stirring for 3 hours. The reaction mixture was concentratedunder reduced pressure, and ethanol (8 mL) and water (2 mL) were addedthereto. Subsequently, triethylamine (2 mL) was added to the resultantmixture, followed by refluxing in an oil bath at 90° C. for 2 hours. Thereaction mixture was concentrated under reduced pressure, and dissolvedin chloroform (200 mL). The resultant solution was washed by a 10%aqueous citric acid solution (50 mL), and the obtained organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. Subsequently, concentrated hydrochloric acid (10 mL)was added to the obtained yellow solid at room temperature, followed bystirring for 10 minutes. The reaction mixture was transferred to aseparatory funnel with 1 mol/L hydrochloric acid (10 mL), followed bywashing chloroform (50 mL×2). The pH of the obtained aqueous layer wasadjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solution undercooling with ice. The pH was adjusted again to 7.4. The resultantmixture was extracted with chloroform (200 mL×3). The combined organiclayer was dried over anhydrous sodium sulfate, filtered, concentratedunder reduced pressure. The obtained residue was recrystallized from anethanol-28% aqueous ammonia mixture (25:1), and dried under reducedpressure, to thereby yield the title compound as yellow crystals (130mg, 45%).

mp:218-220° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δppm:0.15-0.29(2H,m),0.49-0.62(2H,m),0.77-0.88(1H,m),1.51(3H,d,J=6.8Hz),1.47-1.54(1H,m),3.40-3.61(2H,m),3.67-3.73(2H,m),3.86-3.93(1H,m),4.28(1H,d,J=11.5Hz),4.47(1H,d,J=11.2 Hz),4.54-4.61(1H,m),7.51(1H,d,J=14.6 Hz)8.31(1H,s).

IR(ATR)ν cm⁻¹:3043,2983,2875,1705,1618,1525,1444,1396,1350,1323,1279,1209.

MS(EI)m/z:388(M+1)⁺.

Anal.:Calcd for C₂₀H₂₂FN₃O₄.0.25H₂O: C, 61.29; H, 5.79; N, 10.72; F,4.85. Found: C, 61.20; H, 5.65; N, 10.78; F, 4.86.

Referential Example 11(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-carboxylpyrrolidine

(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-formylpyrrolidine(6.00 g, 12.0 mmol) was suspended in tertiary butyl alcohol (16 mL).Subsequently, 2-methyl-2-butene (12.7 mL, 120 mmol) was added to thesuspension, and a suspension of sodium hypochlorite (1.09 g, 12.0 mmol)and sodium dihydrogenphosphate dihydrate (1.87 g, 12.0 mmol) in water(12 mL) was further added thereto, followed by stirring at roomtemperature for 12 hours. Under cooling with ice, 1 mol/L hydrochloricacid (80 mL) was added to the resultant mixture, and the obtainedaqueous layer was extracted with diethyl ether (250 mL×2). The combinedorganic layer was washed sequentially by 5% aqueous sodium thiosulfatesolution (80 mL) and saturated brine (80 mL), dried over anhydroussodium magnesium, filtered, and concentrated under reduced pressure, tothereby yield(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-carboxylpyrrolidineas a colorless oily substance (7.10 g, quantitative amount). The productwas employed, without further purification, in the subsequent step.

¹H-NMR(400 MHz,CDCl₃)δppm:1.28(9H,s),3.25-3.30(1H,m),3.38-3.51(1H,m),3.67-3.78(4H,m),4.53-4.33(1H,m),5.12(2H,s),7.29-7.40(5H,m).

MS(ESI)m/z:265(M-Boc)⁺.

Referential Example 12(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-methoxycarbonylpyrrolidine

The above-obtained crude(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-carboxylpyrrolidine(7.10 g, 12.0 mmol) was dissolved in a diethyl ether-dichloromethanesolvent mixture (2:1) (150 mL). Subsequently, an excessive amount of asolution of diazomethane in diethyl ether (60 mL) was added to thesolution, followed by stirring under cooling with ice for 10 minutes.After completion of the reaction, the reaction mixture was furtherstirred at room temperature for 2 hours, followed by concentration underreduced pressure. The obtained residue was recrystallized from achloroform/n-hexane solvent mixture, and dried under reduced pressure,to thereby yield the title compound as colorless solid (3.37 g, 74%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.43(9H,s),3.19-3.28(1H,m),3.38-3.49(1H,m),3.67-3.79(7H,m),4.49(1H,brs),5.11-5.15(2H,m),7.31-7.40(5H,m).

MS(ESI)m/z:279(M-Boc)⁺.

Referential Example 13(3S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(1-hydroxy-1-methyl)ethylpyrrolidine(4-position Isomer Mixture)

(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-methoxycarbonylpyrrolidine(1.00 g, 2.64 mmol) was dissolved in tetrahydrofuran (20 mL), and asolution of methylmagnesium bromide in tetrahydrofuran (1.00 mol/L, 8.71mL, 8.71 mmol) was added dropwise to the solution under a nitrogenatmosphere at −10° C. over 10 minutes. After stirring of the resultantmixture at the same temperature for 10 minutes, a saturated aqueousammonium chloride solution (20 mL) was added thereto, followed bystirring for 0.5 hours. The obtained aqueous layer was extracted withdiethyl ether (50 mL×2). The obtained organic layer was washed bysaturated brine (40 mL), dried over anhydrous sodium magnesium,filtered, and concentrated under reduced pressure. The obtained residuewas purified through short silica gel column chromatography (ethylacetate:n-hexane=1:2 to 1:1), to thereby yield the title compound(4-position isomer mixture) as a colorless oily substance (1.00 g,quantitative amount). The product was employed, without furtherpurification, in the subsequent step.

¹H-NMR(400 MHz, CDCl₃)δppm:1.24-1.28(3H,m),1.37(3H,s),1.44(9H,s),1.99-2.03(1H,m),2.13-2.21(1H,m),3.41-3.77(4H,m),4.09-4.18(1H,m),5.10-5.18(2H,m),7.29-7.41(5H,m).

MS(ESI)m/z:279(M-Boc)⁺.

Referential Example 14(3S)-1-(Benzyloxycarbonyl)-3-(tert-butoxycarbonyl)amino-4-isopropenylpyrrolidine(4-Position Isomer Mixture)

The above-obtained crude(3S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(1-hydroxy-1-methyl)ethylpyrrolidine(4-position isomer mixture) (1.00 g, 2.64 mmol) was dissolved indichloromethane (20 mL). Subsequently, under cooling with ice,triethylamine (1.47 mL, 10.6 mmol), 4-dimethylaminopyridine (1.29 g,10.6 mmol), and methanesulfonyl chloride (817 μL, 10.6 mmol) were addedto the solution, followed by stirring at room temperature for 12 hours.Under cooling with ice, a saturated aqueous ammonium chloride solution(20 mL) was added to the resultant mixture, and the obtained aqueouslayer was extracted with ethyl acetate (50 mL×2). The combined organiclayer was washed by saturated brine (40 mL), and dried over anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure, and the obtained residue was purified through silicagel column chromatography (ethyl acetate:n-hexane=1:4 to 1:1), tothereby yield the title compound (4-position isomer mixture) as acolorless oily substance (450 mg, 47%).

¹H-NMR(400 MHz,CDCl₃)δ:1.44(9H,s),1.57(3H,d,J=0.5Hz),1.75-1.77(2H,m),3.55(1H,dd,J=5.1,12.0 Hz),3.76(1H,d,J=11.8Hz),3.96(1H,d,J=13.2Hz),4.06-4.16(1H,m),4.63(2H,s),5.15(2H,s),7.34-7.39(5H,m).

MS(ESI)m/z:261(M-Boc)⁺.

Referential Example 15(35)-3-(tert-Butoxycarbonyl)amino-4-isopropylpyrrolidine(4-PositionIsomer Mixture)

A 10% palladium carbon catalyst (300 mg) was added to a solution of(3S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-isopropenylpyrrolidine(300 mg, 832 μmol) in methanol (10 mL), and the resultant mixture wasstirred in a hydrogen atmosphere under an ordinary pressure for 2 hours.After filtration, a 10% palladium carbon catalyst (300 mg) was addedagain to the filtrate, followed by stirring in a hydrogen atmosphereunder an ordinary pressure for 1 hour. After filtration, the filtratewas concentrated under reduced pressure, to thereby yield(3S)-3-(tert-butoxycarbonyl)amino-4-isopropylpyrrolidine as a colorlessamorphous (172 mg, quantitative amount).

MS(ESI+)m/z:229(M+1)⁺.

Example 10(3S)-10-[(3S)-3-Amibo-4-isopropropyl-1-pyrrolidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Isomer A: Compound No. 10-A) and (Isomer B: Compound No. 10-B)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (247 mg, 752 μmol) and triethylamine (126 μL,902 μmol) were added to a solution of(3S)-3-(tert-butoxycarbonyl)amino-4-isopropylpyrrolidine (172 mg, 752μmol) in dimethyl sulfoxide (2 mL), and the resultant mixture wasstirred at room temperature for 20 hours. The reaction mixture wasconcentrated under reduced pressure, and ethanol (50 mL), water (2 mL),and triethylamine (1 mL) were added thereto, followed by refluxing in anoil bath at 100° C. for 4 hours. The reaction mixture was concentratedunder reduced pressure, and dissolved in ethyl acetate (100 mL×2). Thesolution was washed sequentially by a 10% aqueous citric acid solution(100 mL) and saturated brine (50 mL). The obtained organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. Subsequently, concentrated hydrochloric acid (5 mL)was added to the obtained yellow solid under cooling with ice, and themixture was stirred at room temperature for 10 minutes. The reactionmixture was transferred to a separatory funnel with 1 mol/L hydrochloricacid (10 mL), followed by washing with chloroform (100 mL×4). The pH ofthe obtained aqueous layer was adjusted to 12.0 with a 10 mol/L aqueoussodium hydroxide solution under cooling with ice. The pH was adjustedagain to 7.4 with concentrated hydrochloric acid and 1 mol/Lhydrochloric acid, and the resultant mixture was extracted withchloroform (150 mL×3). The combined organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure, to thereby yield a crude mixture of isomer A and isomer B(about 1:4). The crude mixture was subjected to separation andpurification through preparative chromatography(chloroform:methanol:water=7:3:1, developer), recrystallization fromethanol, and drying under reduced pressure, to thereby obtain isomer A(48.5 mg, 17%, a low polar compound) and isomer B (90.5 mg, 31%, a highpolar compound), both being in the form of yellow crystals.

Isomer A;

mp:249-251° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.93(3H,d,J=6.6 Hz),1.00(3H,d,J=6.4Hz),1.52(3H,d,J=6.9 Hz), 1.66-1.86(1H,m),3.34(1H,dd,J=11.0,1.2Hz),3.49-3.54(2H,m),3.73(1H,td,J=10.3,2.7 Hz),4.08(1H,dt,J=10.6,3.9 Hz),4.26(1H,dd,J=11.3,2.5 Hz),4.46(1H,dd,J=11.4,1.8Hz),4.55-4.61(1H,m),7.52(1H,d,J=14.5 Hz),8.31(1H,s).

IR(ATR)ν cm⁻¹:3383,3317,2947,2889,1716,1618,1535,1437,1398,1346,1315,1284,1232.

MS(ESI+)m/z:390(M+1)⁺.

Anal.Calcd for C₂₀H₂₄FN₃O₄.0.25H₂O: C, 60.98; H, 6.27; N, 10.67; F,4.82. Found: C, 61.17; H, 6.19; N, 10.63; F, 4.96.

Isomer B;

mp:202-205° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.93(3H,d,J=6.1 Hz),1.01(3H,d,J=6.4Hz),1.19(1H,t,J=7.1 Hz), 1.50(3H,d,J=6.9Hz),1.76-1.84(2H,m),3.28(1H,q,J=6.7Hz),3.41-3.48(2H,m),3.58-3.68(3H,m),4.32(1H,dd,J=11.4,2.3Hz),4.48(1H,dd,J=11.3,2.0 Hz), 4.55-4.61(1H,m),7.51(1H,d,J=14.2Hz),8.32(1H,s).

IR(ATR)ν cm⁻¹:2958,2871,1720,1616,1576,1522,1444, 1383,1340,1257,1213.

MS(ESI+)m/z:390(M+1)⁺.

Anal:Calcd for C₂₀H₂₄FN₃O₄.0.5EtOH.0.75H₂O: C, 59.21; H, 6.74; N, 9.86;F, 4.46. Found: C, 59.30; H, 6.53; N, 9.99; F, 4.56.

Referential Example 16(3S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-formylpyrrolidine

Dimethyl sulfoxide (5.10 mL, 59.3 mmol) was added to a solution ofoxalylchloride (5.00 mL, 57.3 mmol) in dichloromethane (100 mL), and themixture was stirred at −78° C. for 20 minutes. Subsequently, a solutionof(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-hydroxymethylpyrrolidine(8.00 g, 22.8 mmol) in dichloromethane (85 mL) was added dropwise slowlythereto, followed by stirring at the same temperature for 30 minutes,and further at −43° C. for 1 hour. Subsequently, triethylamine (30 mL)was added thereto, and the temperature of the resultant mixture waselevated to an ice cooling temperature, followed by stirring for 45minutes. Thereafter, a 10% aqueous citric acid solution (100 mL) wasadded to the resultant mixture, followed by stirring for 10 minutes. Theobtained aqueous layer was extracted with dichloromethane (200 mL×2).The combined organic layer was washed by saturated brine (100 mL), anddried over anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure, to thereby yield a crude aldehydeas a pale yellow oilily substance. Subsequently, triethylamine (0.50 mL,3.59 mmol) was added to a solution of the crude aldehyde compound (2.5g, 7.18 mmol) in dichloromethane (10 mL). The resultant mixture wasstirred at room temperature for 24 hours, and triethylamine (0.50 mL,3.59 mmol) was further added thereto, followed by stirring for another24 hours. Thereafter, silica gel (100 g) was added to the reactionmixture, followed by stirring for 24 hours. The reaction mixture wasconcentrated under reduced pressure, and left to stand for 24 hours. Theresidue was purified through silica gel column chromatography (ethylacetate:n-hexane=7:3), to thereby yield the title compound (an isomermixture of (4S)-form and (4R)-form (1:3)) as a colorless oily substance(750 mg, 28%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.42(9×1/4H,s),1.44(9×3/4H,s),2.90-3.93(5H,m),4.40-5.30(4H,m),7.24-7.50(5H,m),9.71(1×3/4H,s),9.77(1H×1/4,s).

MS(ESI)m/z:249(M-Boc)⁺.

Referential Example 17(3S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine

Butyl lithium (3.56 mL, 1.58 M, toluene solution) was added dropwiseunder cooling with ice to a suspension of methyltriphenylphosphoniumbromide (2.15 g, 6.03 mmol) in tetrahydrofuran (20 mL), followed bystirring at room temperature for 20 minutes, and further at 40° C. for 5minutes. Subsequently, a solution of(3S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-formylpyrrolidine[an isomer mixture of (4S)-form:(4R)-form (1:3)] (700 mg, 2.01 mmol) intetrahydrofuran (7.0 mL) was added under cooling with ice to theresultant mixture, followed by stirring at room temperature for 10hours. The reaction mixture was poured to a 10% aqueous citric acidsolution (20 mL), and the resultant mixture was diluted with ethylacetate (100 mL). The obtained aqueous layer was extracted with ethylacetate (100 mL×2). The combined organic layer was washed sequentiallyby saturated sodium hydrogencarbonate (100 mL) and saturated brine (100mL), and dried over anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure. The obtained residuewas purified through silica gel column chromatography (ethylacetate:n-hexane=1:4), to thereby yield the title compound (an isomermixture of (4S)-form and (4R)-form (1:4)) as a colorless oily substance(250 mg, 36%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.43(9H,s),2.50-2.75(1×4/5H,m),2.89-3.02(1×1/5H,m),3.05-3.48(2H,m),3.56-4.01(3H,m),4.25(1×1/5H,s),4.53(1×4/5H,s),5.06-5.30(4H,m),5.65-5.84(1H,m),7.28-7.40(5H,m).

MS(ESI+)m/z:347(M+1)⁺.

Referential Example 18(3S)-3-(tert-Butoxycarbonyl)amino-4-ethylpyrrolidine

A 10% palladium carbon catalyst (15 mg) was added to a solution of(3S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-vinylpyrrolidine[an isomer mixture of (4S)-form:(4R)-form (1:4)] (230 mg, 0.66 mmol) inethanol (8 mL), followed by stirring in a hydrogen atmosphere under anordinary pressure at room temperature for 12 hours. After filtration,the filtrate was concentrated under reduced pressure, to thereby yieldthe title compound (an isomer mixture of (4S)-form and (4R)-form (1:4))as a colorless oily substance (148 mg, 100%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.94(3H,t,J=7.4Hz),1.20-1.80(3H,m),1.44(9H,s),1.90-2.20(1H,m),2.50-2.59(1H,m),2.76(1×4/5H,dd,J=4.4,11.3Hz),2.79(1×1/5H,dd,J=3.1, 11.1Hz),3.12-3.24(2H,m),3.71(1H,s),4.17(1×1/5H,brs),4.77(1×4/5H,brs).

MS(ESI)m/z:215(M+1)⁺.

Example 11(3S)-10-[(3S,4R)-3-Amino-4-ethylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Compound No. 11)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (223 mg, 676 μmol) and triethylamine (113 μL,812 μmol) were added to a solution of(3S)-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine (145 mg, 676 μmol)in dimethyl sulfoxide (3 mL), and the resultant mixture was stirred atroom temperature for 16 hours. Subsequently, water (10 mL) was added tothe reaction mixture, and precipitated crystals were collected throughfiltration. The thus-collected crystals were suspended in a mixture ofethanol (10 mL) and water (2 mL), and triethylamine (3 mL) was addedthereto, followed by refluxing in an oil bath at 90° C. for 5 hours. Thereaction mixture was concentrated under reduced pressure, and dissolvedin chloroform (100 mL). The solution was washed by a 10% aqueous citricacid solution (30 mL). The obtained organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. Subsequently, concentrated hydrochloric acid (5 mL) was addedto the obtained yellow solid under cooling with ice, followed bystirring for 30 minutes. The reaction mixture was transferred to aseparatory funnel with 4 mol/L hydrochloric acid (4 mL), followed bywashing by chloroform (50 mL×7). The pH of the obtained aqueous layeradjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solution undercooling with ice. The pH was adjusted again to 7.4, and the resultantmixture was extracted with chloroform (70 mL×3). The combined organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The obtained residue was purifiedthrough preparative chromatography (chloroform:methanol:water=7:3:1,developer), recrystallized from ethanol, and dried under reducedpressure, to thereby yield the title compound as yellow crystals (80 mg,31%).

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.96(3H,t,J=7.5Hz),1.27-1.38(1H,m),1.51(3H,d,J=6.9Hz),1.65-1.76(1H,m),1.79-1.91(1H,m),3.12(1H,q,J=7.4Hz),3.37-3.48(2H,m),3.71(2H,dd,J=16.3,7.0 Hz),4.32(1H,dd,J=2.5,11.8 Hz),4.48(1H,dd,J=2.2,11.8 Hz),4.55-4.63(1H,m),7.52(1H,d,J=14.0Hz),8.32(1H,s).

mp:189-191° C.

IR(ATR)ν cm⁻¹:2978,2937,2875,1718,1614,1572,1522,1462,1379,1325,1254,1207.

[α]_(D) ^(24.8)−43.52° (c0.125,0.1 mol/L NaOH).

MS(ESI)m/z:376(M+1)⁺.

Anal.:Calcd for C₁₉H₂₂FN₃O₄.0.5H₂O: C, 59.37; H, 6.03; N, 10.93; F,4.94. Found: C, 59.33; H, 5.95; N, 10.95; F, 4.93.

Referential Example 19(3S,4S)-1-Benzyloxycarbonyl-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-ethylpyrrolidine

(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine(1.00 g, 2.87 mmol) was added to a suspension of sodium hydride (250 mg,5.74 mmol) in dimethylformamide (14.4 mL), and the resultant mixture wasstirred under cooling with ice for 1 hour. Subsequently, methyl iodide(715 μL, 11.5 mmol) was added to the reaction mixture under cooling withice, followed by stirring at room temperature for 17 hours. After thereaction mixture was cooled on ice, a saturated aqueous ammoniumchloride solution (30 mL) and water (50 mL) were added thereto, followedby extraction with ethyl acetate (200 mL×2). The combined organic layerwas washed sequentially by water (100 mL) and saturated brine (100 mL),and obtained organic layer was dried over anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure.The obtained residue was purified through silica gel columnchromatography (ethyl acetate:n-hexane=1:4 to 1:2), to thereby yield thetitle compound as a colorless oily substance (989 mg, 95%).

[α]_(D) ^(23.7) +37.58° (c1.40,CHCl₃).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.94(3H,dt,J=6.1,7.3Hz),1.19-1.33(1H,m),1.41-1.55(2H,m),1.46(9H,d,J=3.4Hz),2.17-2.33(1H,m),2.75(3H,s),3.02-3.13(1H,m),3.55-3.77(3H,m),5.09-5.19(2H,m),7.27-7.38(5H,m).

MS(EI)m/z:363(M+1)⁺.

Referential Example 20(3S,4S)-3-(N-tert-Butoxycarbonyl-N-methyl)amino-4-ethylpyrrolidine

A 10% palladium carbon catalyst (95.2 mg) was added to a solution of(3S,4S)-1-benzyloxycarbonyl-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-ethylpyrrolidine(952 mg, 2.63 mmol) in methanol (26 mL), and the resultant mixture wasstirred in a hydrogen atmosphere under an ordinary pressure at roomtemperature for 1 hour. After filtration, the filtrate was concentratedunder reduced pressure, to thereby yield the title compound as acolorless oily substance (600 mg, 100%). The product was employed,without further purification, in the subsequent step.

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.93(3H,t,J=7.4Hz),1.12-1.27(1H,m),1.46(9H,s),1.46-1.50(2H,m),2.03-2.05(1H,m),2.59(1H,t,J=10.4Hz),2.83(3H,s),3.01(1H,dd,J=4.3,12.1 Hz),3.20(1H,dd,J=8.06,11.0Hz),3.23-3.31(1H,m).

MS(EI)m/z:229(M+1)⁺.

Example 127-[(3S,4S)-3-Ethyl-4-methylaminopyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 12)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (142 mg, 394 μmol) and triethylamine (65.8μL, 472 μmol) were added to a solution of(3S,4S)-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-ethylpyrrolidine (99.0mg, 434 μmol) in dimethyl sulfoxide (2 mL), and the resultant mixturewas stirred at room temperature for 24 hours. The reaction mixture wasconcentrated, and ethanol (20 mL) and water (1 mL) were added theretofor dissolving. Subsequently, triethylamine (2 mL) was added to thesolution, followed by refluxing in an oil bath at 90° C. for 2 hours.The reaction mixture was concentrated under reduced pressure, anddissolved in chloroform (100 mL). The solution was washed by a 10%aqueous citric acid solution (30 mL). The obtained organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. Subsequently, concentrated hydrochloric acid (4 mL)was added so as to dissolve the obtained yellow solid at roomtemperature, and water (20 mL) was added to the solution, followed bywashing by chloroform (80 mL×3). The pH of the obtained aqueous layerwas adjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solutionunder cooling with ice. The pH was adjusted to 7.4. The resultantmixture was extracted with chloroform (80 mL×2). The combined organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The obtained residue wasrecrystallized from ethanol, and dried under reduced pressure, tothereby yield the title compound as yellow crystals (77.0 mg, 45%).

mp:143-145° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.97(3H,t,J=7.3Hz),1.30-1.66(4H,m),2.21-2.30(1H,m),2.34(3H,s),3.23-3.30(1H,m),3.48-3.59(5H,m),3.63-3.80(2H,m),4.00-4.07(1H,m),4.98(1H,d,J=64.5Hz),7.65(1H,d,J=14.6 Hz),8.43(1H,s).

IR(ATR)ν cm⁻¹:3045,2964,2871,1728,1649,1614,1577,1493,1439,1383,1346,1311,1290,1255,1201.

MS(EI)m/z:422(M+1)⁺.

Anal.:Calcd for C₂₁H₂₅F₂N₃O₄.0.75H₂O: C, 57.99; H, 6.14; N, 9.66; F,8.74. Found: C, 57.83; H, 6.05; N, 9.38; F, 8.58.

Example 13(3S)-10-[(3S,4S)-3-Ethyl-4-methylaminopyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (Compound No. 13)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (118 mg, 358 μmol) and triethylamine (60.0μL, 430 μmol) were added to a solution of(3S,4S)-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-ethylpyrrolidine (90.0mg, 394 μmol) in dimethyl sulfoxide (2 mL), and the resultant mixturewas stirred at room temperature for 15 hours. The reaction mixture wasconcentrated under reduced pressure, and ethanol (19 mL) and water (1mL) were added thereto for dissolving. Subsequently, triethylamine (2mL) was added to the solution, followed by refluxing in an oil bath at90° C. for 4 hours. The reaction mixture was concentrated under reducedpressure, and dissolved in chloroform (100 mL). The solution was washedby a 10% aqueous citric acid solution (50 mL). The obtained organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Thereafter, concentratedhydrochloric acid (6 mL) was added to the obtained yellow solid, and theresultant mixture was transferred to a separatory funnel with water (20mL), followed by washing by chloroform (100 mL×2). The pH of theobtained aqueous layer was adjusted to 11.0 with a 10 mol/L aqueoussodium hydroxide solution under cooling with ice. The pH was adjustedagain to 7.4. The resultant mixture was extracted with chloroform (100mL×3). The combined organic layer was dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The obtainedresidue was recrystallized from ethanol, and dried under reducedpressure, to thereby yield the title compound as pale yellow crystals(89.0 mg, 64%).

mp:211-213° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.95(3H,t,J=7.2Hz),1.30-1.43(1H,m),1.46-1.55(1H,m),1.49(3H,d,J=6.6Hz),2.10-2.23(1H,m),2.31(3H,s),3.14-3.23(1H,m),3.49-3.65(3H,m),3.74-3.85(1H,m),4.26(1H,d,J=11.7Hz),4.43(1H,d,J=11.2 Hz),4.51-4.60(1H,m),7.46(1H,d,J=14.4Hz),8.29(1H,s).

IR(ATR)ν cm⁻¹:2873,1705,1620,1525,1464,1444,1394,1352,1321,1292,1207,1184.

MS(EI)m/z:390(M+1)⁺.

Anal.:Calcd for C₂₀H₂₄FN₃O₄: C, 61.68; H, 6.21; N, 10.79; F, 4.88.Found: C, 61.27; H, 6.16; N, 10.69; F,4.85.

Referential Example 21(3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(phenylsulfanil)methylpyrrolidine

A solution of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-hydroxymethylpyrrolidine(7.55 g, 21.55 mmol), diphenyldisulfide (6.55 g, 30 mmol), andtributylphosphine (9.97 mL, 40 mmol) in tetrahydrofuran (100 mL) wasrefluxed under a nitrogen atmosphere for 1 hour. The temperature of theresultant mixture was cooled to room temperature. Subsequently, a 1mol/L aqueous sodium hydroxide solution was added to the mixture,followed by extraction with diethyl ether (200 mL×3). The combinedorganic layer was washed sequentially with water (100 mL) and saturatedbrine (100 mL). The thus-washed organic layer was dried over anhydroussodium magnesium, filtered, and concentrated under reduced pressure. Theobtained residue was purified through silica gel column chromatography(n-hexane: ethyl acetate=4:1), to thereby yield the title compound as acolorless oily substance (7.97 g, 83.6%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.45(9H,s),2.44-2.58(1H,m),2.70-2.80(1H,m),3.16-3.22(2H,m),3.37-3.44(1H,m),3.59(1H,dd,J=5.5,11.5Hz),3.67-3.78(1H,m),4.25-4.32(1H,m),4.64(1H,br),5.11(2H,s),7.18-7.24(1H,m),7.26-7.36(9H,m).

IR(ATR)ν cm⁻¹:3319,2976,1684,1522,1417,1363,1243,1160,737,692.

HRMS(EI)m/z:Calcd for C₂₄H₃₀O₄N₂S: 442.1926. Found: 442.1904.

Referential Example 22(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-methylpyrrolidine

A suspension of(3S,4S)-1-benzyloxycarbonyl-3-(tert-butoxycarbonyl)amino-4-(phenylsulfanil)methylpyrrolidine(7.97 g, 18.01 mmol) and Raney nickel (R100) (50 mL) in ethanol (150 mL)was stirred in a hydrogen atmosphere under an ordinary pressure at anexternal temperature of 50° C. for 18 hours. The temperature of theresultant mixture was cooled to room temperature. Thereafter, insolublematter was removed through filtration by use of Celite, and the filtratewas concentrated under reduced pressure. The obtained residue wasdissolved in ethanol (100 mL), and a 10% palladium carbon catalyst (2.0g) was added to the solution, followed by stirring in a hydrogenatmosphere under an ordinary pressure at room temperature for 3 hours.After insoluble matter was removed through filtration by use of Celite,the filtrate was concentrated under reduced pressure. The obtainedresidue was dissolved in diethyl ether. The solution was washed by a 1mol/L aqueous sodium hydroxide solution, dried over anhydrous sodiumsulfate, filtered, concentrated under reduced pressure, and dried underreduced pressure, to thereby yield the title compound as colorlesscrystals (2.602 g, 72.1%).

¹H-NMR (400 MHz, CDCl₃)δ ppm:0.97(3H,d,J=7.0Hz),1.45(9H,s),1.79(1H,br),2.21-2.28(1H,m),2.48(1H,dd,J=8.5,10.5),2.70(1H,dd,J=4.5,11.0Hz),3.15(1H,dd,J=8.0,10.5 Hz),3.25(1H,dd,J=6.5,11.0Hz),4.10(1H,br),4.63(1H,br).

IR(ATR)ν cm⁻¹:3365,3217,2956,1678,1520,1365,1244,1167, 1061,906,627.

MS (FAB)m/z (%): 201(93,M⁺+1), 145 (100,M⁺−55), 101 (8,M⁺−99).

HRMS (FAB)m/z: Calcd for C₁₀H₂₁O₂N₂: 201.1603. Found: 201.1602.

Referential Example 237-[(3S,4S)-3-Amino-4-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylicacid.hydrochloride (Comparative Compound A)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (1.44 g, 4.00 mmol) and triethylamine (2 mL)were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidine (841 mg, 4.20mmol) in dimethyl sulfoxide (6 mL), and the resultant mixture wasstirred at room temperature for 16 hours. The reaction mixture wasconcentrated under reduced pressure. Subsequently, water was added tothe obtained residue, and precipitated crystals were collected throughfiltration. Thereafter, 90% ethanol (100 mL) and triethylamine (20 mL)were added to the obtained crystals, followed by refluxing for 3 hours.The temperature of the resultant mixture was cooled to room temperature.The reaction mixture was concentrated under reduced pressure. Theobtained residue was dissolved in chloroform (200 mL). The solution waswashed sequentially by a 10% aqueous citric acid solution (100 mL) andsaturated brine (50 mL), dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The obtained residue wasdissolved in concentrated hydrochloric acid, and the solution was washedby chloroform (50 mL). Subsequently, water was added thereto, andprecipitated crystals were collected through filtration. The obtainedcrystals were recrystallized from ethanol, to thereby yield the titlecompound as yellow crystals (900 mg, 50.3%).

mp:246-247° C.

¹H-NMR(400 MHz,D₂O)δ ppm:1.12(3H,d,J=7.0Hz),1.35-1.48(1H,m),1.48-1.60(1H,m),2.63-2.70(1H,m),3.42-3.48(1H,m),3.45(3H,s),3.60-3.63(1H,m),3.70(1H,t,J=9.0Hz),3.91-3.99(3H,m),4.86-5.04(1H,m),6.69(1H,d,J=14.0 Hz),8.48(1H,s).

IR(ATR)ν cm⁻¹:2968,2879,2805,1701,1618,1510,1452,1317,1176,1043,927,804.

Anal.:Calcd for C₁₈H₂₀FN₃O₄.HCl.H₂O: C, 50.95; H, 5.40; N, 9.38; F,8.48; Cl, 7.92. Found: C, 51.02; H, 5.69; N, 9.14; F, 8.32; Cl, 7.77.

Referential Example 24(3S)-10-[(3S,4S)-3-Amino-4-methylpyrrolidin-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid.hydrochloride (Comparative Compound B)

(3S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid difluoroboran complex (1.97 g, 6.00 mmol) and triethylamine (5 mL)were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidine (1.30 g, 6.50mmol) in dimethyl sulfoxide (25 mL), and the resultant mixture wasstirred at room temperature for 7 days. The reaction mixture wasconcentrated under reduced pressure. Subsequently, water was added tothe residue, and precipitated crystals were collected throughfiltration. Thereafter, 90% ethanol (100 mL) and triethylamine (20 mL)were added to the collected crystals, followed by refluxing for 3 hours.The temperature of the resultant mixture was cooled to room temperature.The reaction mixture was concentrated under reduced pressure. Theobtained residue was dissolved in chloroform (300 mL). The solution waswashed sequentially by a 10% aqueous citric acid solution (200 mL) andsaturated brine (100 mL), dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The obtained residue wasdissolved in concentrated hydrochloric acid. The solution was dilutedwith water, and washed by chloroform (50 mL). The reaction mixture wasconcentrated under reduced pressure, and precipitated crystals wererecrystallized from ethanol and a small amount of water, to therebyyield the title compound as yellow crystals (1.40 g, 57.5%).

mp:249-259° C.

¹H-NMR(400 MHz,D₂O)δ ppm:1.10(3H,d,J=7.0 Hz),1.45(3H,d,J=7.0Hz),2.58-2.67(1H,m),3.55-3.61(1H,m),3.65-3.75(1H,m),3.82-3.86(1H,m),4.06-4.12(1H,m),4.23(1H,dd,J=2.0,12.5Hz), 4.37(1H,dd,J=2.0,11.5 Hz),4.55-4.62(1H,m), 6.83(1H,d,J=14.0Hz),8.42(1H,s).

IR(ATR)ν cm⁻¹:3217,2993,2808,1701,1620,1523,1458,1360,1277,1068,987,802.

[α]_(D)−177.20° (c0.660,H₂O).

Anal.: Calcd for C₁₈H₂₀FN₃O₄.HCl.0.25H₂O: C, 53.73; H, 5.39; N, 10.44;F, 4.72; Cl, 8.81. Found: C, 53.76; H, 5.37; N, 10.48; F, 4.94; Cl,8.89.

Referential Example 25 Ethyl4-[(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-2,5-difluoro-3-methylbenzoate

A solution of (3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidine(1.80 g, 8.97 mmol), ethyl 3-methyl-2,4,5-trifluorobenzoate (2.18 g,10.0 mmol), and 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU) (2.24 mL, 15mmol) in dimethyl sulfoxide (20 mL) was stirred under a nitrogenatmosphere at an external temperature of 60° C. for 70 hours. Thetemperature of the mixture was cooled to room temperature.

The resultant mixture was poured to a 10% aqueous citric acid solution,followed by extraction with ethyl acetate (200 mL×3), washing by water(200 mL), saturated aqueous sodium hydrogencarbonate (150 mL), andsaturated brine (150 mL), and dried over anhydrous sodium magnesium.After filtration, the filtrate was concentrated under reduced pressure.The obtained residue was purified through silica gel columnchromatography (n-hexane:ethyl acetate=6:1), to thereby yield the titlecompound as colorless crystals (2.51 g, 70.2).

¹H-NMR(400 MHz,CDCl₃)δ ppm:1.07(3H,d,J=7.0 Hz),1.38(3H,t,J=7.0Hz),1.47(9H,s),2.20(3H, d,J=2.5 Hz),2.50-2.57(1H,m),3.06-3.12(1H,m),3.16(1H,dd,J=3.0,10.0 Hz),3.39-3.44(1H,m),3.73-3.77(1H,m),4.27-4.33(1H,m),4.36(2H,q,J=7.0 Hz), 4.71(1H,br),7.42(1H,dd,J=7.0,13.0Hz).

MS(ESI)m/z:399(M⁺+1).

Referential Example 264-[(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-2,5-difluoro-3-methylbenzoicacid

A 1 mol/L aqueous sodium hydroxide solution (15 mL, 15 mmol) was addedunder cooling with ice to a solution of ethyl4-[(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-2,5-difluoro-3-methylbenzoate(2.51 g, 6.30 mmol) in ethanol (20 mL), followed by stirring at roomtemperature for 20 hours. The resultant mixture was neutralized with 1mol/L hydrochloric acid under cooling with ice, and ethanol was removedunder reduced pressure. Subsequently, 1 mol/L hydrochloric acid wasadded to the residue, and the mixture was extracted with ethyl acetate(200 mL×2), followed by drying over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, anddried under reduced pressure (solvent), to thereby yield the titlecompound as a yellow oily substance (2.20 g, quantitative amount). Theproduct was employed, without further purification, in the subsequentstep.

¹H-NMR (400 MHz, CDCl₃)δ ppm:1.08(3H,d,J=6.5Hz),1.47(9H,s),2.21(3H,d,J=2.0Hz),2.50-2.56(1H,m),3.13-3.25(2H,m),3.43-3.49(1H,m),3.81(1H,dd,J=6.0,8.5Hz),4.28-4.34(1H,m),4.73(1H,br),7.48(1H,dd,J=7.0,13.0 Hz).

MS(ESI)m/z:371(M⁺1).

Referential Example 27 Ethyl3-[((3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl)-2,5-difluoro-3-methylphenyl]-3-oxopropionate

1,1′-carbonyldiimidazole (1.46 g, 9.00 mmol) was added to a solution of4-[(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-2,5-difluoro-3-methylbenzoicacid (2.20 g, 6.30 mmol) in tetrahydrofuran (30 mL), and the mixture wasstirred at room temperature for 3 hours. Separately, a magnesium saltwas prepared potassium ethyl malonate (3.40 g, 20.0 mmol), magnesiumchloride (2.38 g, 25.0 mmol), and triethylamine (4.18 mL, 30.0 mmol),and the salt was dissolved in ethyl acetate (40 mL). The reactionmixture was added dropwise to the solution under cooling with ice,followed by stirring at an external temperature of 50° C. for 16 hours.Under cooling with ice, a 10% aqueous citric acid solution was addedthereto, and the resultant mixture was extracted with ethyl acetate (200mL×2), washed by saturated sodium hydrogencarbonate (100 mL) andsaturated brine (100 mL), and dried over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe obtained residue was purified through silica gel columnchromatography (n-hexane:ethyl acetate=4:1), to thereby yield the titlecompound (2.58 g, 93.0%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:1.07(3H,d,J=7.0Hz),1.24-1.38(3H,m),1.47(9H,s),2.20(3H,d,J=2.5Hz),2.49-2.55(1H,m),3.02-3.24(2H,m),3.37-3.50(1H,m),3.69-3.84(1H,m),3.91-3.93(1.4H,m),4.18-4.30(3H,m),5.83(0.3H,s),7.35-7.48(1H,m),12.68(0.3H,s).

MS(ESI)m/z:441(M⁺+1).

Referential Example 28 Ethyl7-[(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate

A solution of ethyl3-[((3S,4S)-3-(tert-butoxycarbonyl)amino-4methylpyrrolidin-1-yl)-2,5-difluoro-3-methylphenyl]-3-oxopropionate(2.58 g, 5.86 mmol) and N,N-dimethylformamide dimethylformamidedimethylacetal (3.99 mL, 30.0 mmol) in benzene (40 mL) was refluxed for4 hours. The temperature of the reaction mixture was cooled to roomtemperature. The reaction mixture was concentrated under reducedpressure. The obtained residue was dissolved in dichloromethane (20 mL),and cyclopropylamine (693 μL, 10.0 mmol) was added dropwise to thesolution under cooling with ice, followed by stirring at roomtemperature for 18 hours. The reaction mixture was concentrated underreduced pressure. The obtained residue was dissolved in 1,4-dioxane (30mL), and 55% oily sodium hydride (436 mg, 10.0 mmol) was added to thesolution at room temperature, followed by stirring for 2 hours.Subsequently, the resultant mixture was poured to an ice-coolingsaturated aqueous ammonium chloride solution. The resultant mixture wasextracted with ethyl acetate (200 mL×2), washed by saturated brine (100mL), and dried over anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure. The obtained residuewas purified through silica gel column chromatography(chloroform:methanol=25:1), to thereby yield the title compound (957 mg,33.4%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.82-0.95(2H,m),1.10(3H,d,J=7.0Hz),1.13-1.23(2H,m),1.41(3H,t,J=7.0Hz),1.47(9H,s),2.55-2.70(4H,m),3.17-3.24(2H,m),3.48-3.53(1H,m),3.82(1H,ddd,J=1.5,5.5,10.0Hz),3.93(1H,ddd,J=3.5,8.0, 14.0Hz),4.32-4.42(3H,m),4.74(1H,br),7.91(1H,d,J=13.5 Hz),8.63(1H,s).

MS(ESI)m/z:488(M⁺+1).

Referential Example 297-[(3S,4S)-3-(tert-Butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Comparative Compound C)

A 1 mol/L aqueous sodium hydroxide solution (5 mL, 5 mmol) was addedunder cooling with ice to ethyl7-[(3S,4S)-3-(tert-butoxycarbonyl)amino-4-methylpyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate(957 mg, 1.96 mmol) in ethanol (10 mL), and the mixture was stirred atroom temperature for 3 hours. The resultant mixture was neutralized with1 mol/L hydrochloric acid under cooling with ice, and ethanol wasremoved under reduced pressure. Subsequently, 1 mol/L hydrochloric acidwas added thereto, followed by extraction with ethyl acetate (100 mL×3).The ethyl acetate layer was washed by saturated brine (100 mL), anddried over anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The obtained residue was dissolvedin concentrated hydrochloric acid (5 mL), and the solution was washed bychloroform (50 mL). The pH of the resultant mixture was adjusted to 7.8with an aqueous sodium hydroxide solution, followed by extraction withchloroform (100 mL×3) and drying over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe obtained residue was recrystallized from ethanol, to thereby yieldthe title compound as pale yellow crystals (420 mg, 58.2%).

¹H-NMR(400 MHz,0.1 mol/L NaOD)δ ppm:0.71-0.85(2H,m),1.05(3H,d,J=7.0Hz),1.09-1.21(2H,m),2.31-2.41(1H,m),2.45(3H,s),3.23(1H,dd,J=3.5,10.0Hz),3.29-3.34(1H,m),3.43-3.48(2H,m),3.75-3.80(1H,m),4.05-4.10(1H,m),7.61(1H,d,J=14.5Hz),8.55(1H,s).

IR(ATR)ν cm⁻¹:2964,2812,1724,1612,1570,1435,1346,1317, 1196,1039,820.

Anal.:Calcd for C₁₉H₂₂FN₃O₃0.5H₂O: C, 61.94; H, 6.29; N, 11.41. Found:C, 62.05; H, 6.03; N, 11.42.

Referential Example 307-[(3S,4S)-3-Amino-4-ethylpyrrolidine-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Comparative Compound D)

1-Cyclopropyl-6,7-difluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (486 mg, 1.48 mmol) and triethylamine (309μL, 2.22 mmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl)amino-4-ethylpyrrolidine (611 mg, 2.85mmol) in sulfolane (4 mL), and the resultant mixture was stirred in anoil bath at 33° C. for 62 hours. Subsequently, cold water (100 mL) waspoured to the reaction mixture, and a precipitated solid was collectedthrough filtration, followed by washing by water (10 mL×3). Ethanol (100mL), water (5 mL), and triethylamine (2 mL) were added to the obtainedsolid, followed by refluxing in an oil bath at 100° C. for 2 hours. Thereaction mixture was concentrated under reduced pressure. Subsequently,a 10% aqueous citric acid solution (50 mL) was added thereto, and themixture was extracted with chloroform (100 mL×2). The obtained organiclayer was washed by saturated brine (50 mL), and dried over anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure. Thereafter, concentrated hydrochloric acid (10 mL) wasadded to the obtained yellow solid (800 mg) under cooling with ice, andthe resultant mixture was stirred at room temperature for 20 minutes.The reaction mixture was washed by chloroform (50 mL×3). The pH of theobtained aqueous layer was adjusted to 12.0 with a 10 mol/L aqueoussodium hydroxide solution under cooling with ice. The pH was adjustedagain to 7.4 with concentrated hydrochloric acid and 1 mol/L aqueoushydrochloric acid, and the mixture was extracted with chloroform (200mL×7). The combined organic layer was dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The obtainedresidue was recrystallized from ethanol, and dried under reducedpressure, to thereby yield the title compound as pale yellow crystals(367 mg, 58%).

mp:148-150° C.

¹H-NMR(400 MHz,0.1 mol/L NaOD)δppm:0.72-0.79(1H,m),0.84-0.90(1H,m),0.97(3H,t,J=7.5Hz),1.07-1.14(1H,m),1.19(3H,t,J=7.1 Hz),1.21-1.26(1H,m),1.46(1H,q,J=7.1Hz),1.53(1H,q,J=7.1 Hz),2.16-2.23(1H,m),2.51(3H,s),3.20(1H,d,J=10.0Hz),3.39(1H,t,J=8.5 Hz), 3.45-3.53(2H,m),3.66(2H,q,J=7.1Hz),3.90-3.94(1H,m),4.08-4.14(1H,m),7.64(1H,d,J=14.5 Hz),8.56(1H,s).

IR(ATR)ν cm⁻¹:3228,2966,2920,2877,1726,1635,1570,1554,1512,1468,1442,1379,1346,1306.

Anal.:Calcd. for C₂₅H₃₂FN₃O₅.1.0CH₃CH₂OH.0.25H₂O: C, 62.32; H, 7.25; N,9.91; F, 4.48. Found: C, 62.04; H, 7.26; N, 9.90; F, 4.65.

Example 147-[(3S,4S)-3-Amino-4-ethylpyrrolidine-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 14)

A 10% palladium carbon catalyst (27 mg) was added to a solution of(3S,4S)-1-(benzyloxycarbonyl)-3-(tert-butoxycarbonylamino)-4-ethylpyrrolidine(270 mg, 775 μmol) in methanol (7.75 mL), and the resultant mixture wasstirred under a hydrogen atmosphere at room temperature for 15 hours.After filtration, the filtrate was concentrated under reduced pressure.The obtained residue was dissolved in dimethyl sulfoxide (3 mL), and7-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (216 mg, 775 μmol) and triethylamine (130 μL, 930 μmol) were addedto the solution, followed by stirring in an oil bath at 65° C. for 8days. The reaction mixture was concentrated under reduced pressure, andthe residue was dissolved in ethyl acetate (50 mL). The solution waswashed sequentially with a 10% aqueous citric acid solution (30 mL),water (30 mL×2), and saturated brine (30 mL), followed by drying overanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. Subsequently, concentratedhydrochloric acid (2 mL) was added to the obtained residue at roomtemperature, and the resultant mixture was transferred to a separatoryfunnel with water (20 mL), followed by washing by chloroform (30 mL×2).The pH of the obtained aqueous layer was adjusted to 12.0 with a 10mol/L aqueous sodium hydroxide solution under cooling with ice. The pHwas adjusted again to 7.4, and the resultant mixture was extracted withchloroform (100 mL×3). The combined organic layer was dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure. The obtained residue was recrystallized from hot ethanol, anddried under reduced pressure, to thereby yield the title compound aspale yellow crystals (145 mg, 50.1%).

mp:232-234° C.

¹H-NMR(400 MHz,0.1N-NaOD)δ ppm:0.96(3H,t,J=7.4Hz),1.13-1.29(1H,m),1.41-1.66(3H,m),2.07-2.23(1H,m),2.34(3H,s),3.06(1H,d,J=10.3Hz),3.19(1H,t,J=8.7 Hz), 3.43-3.60(2H,m),3.77(1H,dd,J=4.6,10.3Hz),3.99-4.04(1H,m),5.03(1H,d,J=65.2 Hz),6.98(1H,d,J=9.0Hz),7.95(1H,d,J=9.0 Hz),8.42(1H,d,J=3.7 Hz).

Anal.:Calcd for C₂₀H₂₄FN₃O₃: C, 64.33; H, 6.48; F, 5.09; N, 11.25.Found: C, 64.01; H, 6.55; F, 4.95; N, 10.89.

MS(ESI)m/z:374(M+1)⁺

IR(ATR):3386,3037,2962,2914,2856,1705,1616,1550,1514,1462,1435,1379,1350,1327 cm⁻¹

Example 157-[(3S,4S)-3-Ethyl-4-methylaminopyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 15)

6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid difluoroboran complex (100 mg, 290 μmol) and triethylamine (48.5μL, 348 μmol) were added to a solution of(3S,4S)-3-(tert-butoxycarbonyl-N-methylamino)-4-ethylpyrrolidine (410mg, 1.80 mmol) in sulfolane (1.5 mL), and the resultant mixture wasstirred at 45° C. for 5 days. Ethanol (20 mL) and water (5 mL) wereadded thereto, and triethylamine (5 mL) was added to the reactionmixture, followed by refluxing in an oil bath at 90° C. for 3 hours. Thereaction mixture was concentrated under reduced pressure, and dissolvedin ethyl acetate (200 mL). The solution was washed sequentially with a10% aqueous citric acid solution (50 mL), water (50 mL×6), and saturatedbrine (50 mL). The obtained organic layer was dried over anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure. The obtained yellow oily substance was purifiedthrough preparative TLC (methanol:chloroform=1:10). Thereafter,concentrated hydrochloric acid (6 mL) was added at room temperature soas to dissolve the obtained yellow oily substance. The solution waswashed with chloroform (50 mL×2). The pH of the obtained aqueous layerwas adjusted to 12.0 with a 10 mol/L aqueous sodium hydroxide solutionunder cooling with ice. The pH was adjusted again to 7.4. The resultantmixture was extracted with chloroform (100 mL×2). The combined organiclayer was dried over anhydrous sodium sulfate, and the solvent wasremoved under reduced pressure. The obtained residue was recrystallizedfrom hot ethanol, and dried under reduced pressure, to thereby yield thetitle compound as pale yellow crystals (28.0 mg, 23.8%).

mp:223-225° C.

¹H-NMR(400 MHz,0.1N-NaOD)δ ppm:0.97(3H,t,J=7.4Hz),1.22-1.68(4H,m),2.24-2.34(1H,m),2.32(3H,s),2.47(3H,s),3.24-3.30(1H,m),3.35-3.57(3H,m),3.80-3.88(1H,m),4.05-4.12(1H,m),5.01(1H,d,J=63.4Hz),7.67(1H,d,J=14.4 Hz),8.44(1H,d, J=2.9 Hz).

Anal.:Calcd for C₂₁H₂₅F₂N₃O₃: C, 62.21; H, 6.22; F, 9.37; N, 10.36.Found: C, 61.97; H, 6.31; F, 9.50; N, 10.11.

MS(ESI)m/z:406(M+1)⁺

IR(ATR):3319,2939,2877,2800,1724,1610,1504,1427,1346,1313,1271,1225,1201,1173,1136,1105 cm⁻¹

Referential Example 314-Isopropylidene-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

Methylmagnesium bromide in tetrahydrofuran (1 mol/L, 1.21 L, 1.21 mol)was added under cooling with ice to a solution of(3S)-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylic acid methylester (100 g, 404 mmol) in tetrahydrofuran (1.20 L), and the resultantmixture was stirred at 0° C. for 30 minutes. Subsequently, a saturatedaqueous ammonium chloride solution (1.00 L) was added to the reactionmixture at the same temperature. The resultant mixture was extractedwith ethyl acetate (1.00 L×2), followed by extraction with saturatedbrine (700 mL). The organic layer was dried over anhydrous sodiumsulfate, and the solvent was removed under reduced pressure, to therebyobtain crude4-(1-hydroxy-1-methyl-ethyl)-2-oxo-1-[(1R)-phenylethyl]pyrrolidine. Theproduct was dissolved in dichloromethane (1.00 L), and triethylamine(225 mL, 1.62 mol) and 4-dimethylaminopyridine (197 g, 1.62 mol) wereadded to the solution. Thereafter, methanesulfonyl chloride (125 mL,1.62 mol) was added dropwise thereto under cooling with ice. The mixturewas stirred at room temperature for 13 hours. Subsequently, a saturatedaqueous ammonium chloride solution (1.00 L) was added to the reactionmixture under cooling with ice. The resultant mixture was extracted withethyl acetate (1.00 L×2), followed by washing by saturated brine (700mL). The obtained organic layer was dried over anhydrous sodium sulfate,and the solvent was removed under reduced pressure. The obtained residuewas purified through silica gel column chromatography (n-hexane:ethylacetate=4:1 to 1:1), to thereby yield the title compound as colorlesssolid (66.0 g, 71%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:1.53(3H,s),1.56(3H,d,J=7.3Hz),1.62(3H,s),3.06-3.10(2H,m),3.52(1H,d,J=13.4 Hz),3.89(1H,d,J=13.4Hz),5.62(1H,q, J=7.2 Hz),7.25-7.38(5H,m).

Referential Example 32(4R&S)-4-Isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

A 10% palladium carbon catalyst (M, water content: 52.8%, 50.0 g) wasadded to a solution of4-isopropylidene-2-oxo-1-[(1R)-phenylethyl]pyrrolidine (52.0 g, 225mmol) in ethanol (1.00 L), and the resultant mixture was stirred in ahydrogen atmosphere at 40° C. for 16 hours. The catalyst was removedthrough filtration (washing by ethanol), and the filtrate wasconcentrated under reduced pressure. The residue was purified throughsilica gel column chromatography (n-hexane:ethyl acetate=3:1 to 1:1), tothereby yield the title compound as a colorless syrupy substance (48.0g, 92%).

¹H-NMR(400 MHz,CDCl₃)δppm:0.77-0.91(6H,m),1.41-1.57(1H,m),1.50-1.53(3H,m),1.89-2.21(2H,m),2.47-2.62(1H,m),2.95-3.07(2H,m),5.46-5.55(1H,m),7.26-7.36(5H,m).

Referential Example 33(3R,4S&3S,4R)-3-Hydroxy-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

Triethyl phosphite (38.9 mL, 227 mmol) was added to a solution of(4R&S)-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine (43.8 g, 189mmol) in diethyl ether (900 mL). Subsequently, lithium diisopropylamidein tetrahydrofuran (2 mol/L, 142 mL, 284 mmol) was added dropwise to theresultant mixture at −90° C., followed by stirring at −80° C. for 20minutes. Thereafter, oxygen was fed into the mixture at −90° C. for 1hour. A saturated aqueous ammonium chloride solution (1.00 L) was addedto the reaction mixture at the same temperature. The resultant mixturewas extracted with ethyl acetate (1.00 L×2), followed by washing withwater (1.00 L) and saturated brine (700 mL). The obtained organic layerwas dried over anhydrous sodium sulfate, and the solvent was removedunder reduced pressure. The obtained residue was recrystallized fromchloroform-n-hexane, to thereby yield the title compound as colorlesssolid (14.1 g, 30%, a mixture of (3R,4S) form and (3S,4R) form (about5:1)).

¹H-NMR(400 MHz,CDCl₃)δppm:0.81-0.88(3H,m),0.99-1.06(3H,m),1.52-1.57(3H,m),1.70-1.90(2H,m),2.88(1H,t,J=9.4Hz),3.02(1H,t,J=9.0 Hz),3.10-3.19(1H,m),4.08(1H,dd,J=8.9,1.8Hz),5.46(1H,q,J=7.2 Hz),7.25-7.38(5H,m).

Referential Example 34(3S,4S)-3-Azide-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

Triethylamine (17.5 mL, 125 mmol) was added to a solution of(3R,4S&3S,4R)-3-hydroxy-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine(14.1 g, 57.0 mmol) in dichloromethane (280 mL). Subsequently,methanesulfonyl chloride (8.82 mL, 114 mmol) was added dropwise to theresultant mixture under cooling with ice. The mixture was stirred at thesame temperature for 30 minutes. Thereafter, a saturated aqueousammonium chloride solution (500 mL) was added to the reaction mixtureunder cooling with ice, followed by extraction with ethyl acetate (500mL×2), and washing by water (200 mL) and saturated brine (200 mL). Theobtained organic layer was dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. The obtained residue wasdissolved in N,N-dimethylformamide (150 mL). Subsequently, sodium azide(9.26 g, 143 mmol) was added to the solution. The resultant mixture wasstirred at 60° C. for 12 hours, and further at room temperature for 48hours. Thereafter, water (500 mL) was added to the reaction mixtureunder cooling with ice, followed by extraction with ethyl acetate (500mL×2). The ethyl acetate layer was washed by water (500 mL×2) andsaturated brine (500 mL). The obtained organic layer was dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure. The obtained residue was purified through silica gel columnchromatography (n-hexane:ethyl acetate=9:1 to 6:1 to 1:1), to therebyyield the title compound as a colorless syrupy substance (11.9 g, 77%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.81(3H,d,J=6.3 Hz),0.97(3H,d,J=6.1Hz),1.56(3H,d,J=7.1 Hz), 1.76-1.89(2H,m),2.96(1H,dd,J=9.6,7.2 Hz),3.04(1H,t,J=9.0 Hz),4.06(1H,d,J=6.3 Hz),5.47(1H,q,J=7.2Hz),7.26-7.38(5H,m).

Referential Example 35(3S,4S)-3-(tert-Butoxycarbonylamino)-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

A 10% palladium carbon catalyst (M, water content: 52.8%, 1.20 g) wasadded to a solution of(3S,4S)-3-azide-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine (11.9g, 43.7 mmol) in ethanol (500 mL), and the resultant mixture was stirredin a hydrogen gas atmosphere for 1 hour. Subsequently,di-tert-butyldicarbonate (19.1 g, 87.4 mmol) was added thereto. Thereaction mixture was further stirred in a hydrogen gas atmosphere for 14hours. Thereafter, the catalyst was removed through filtration (washingby ethanol), and the filtrate was concentrated under reduced pressure.The obtained residue was purified through silica gel columnchromatography (n-hexane:ethyl acetate=4:1), to thereby yield the titlecompound as colorless solid (13.9 g, 92%).

¹H-NMR(400 MHz,CDCl3)δ ppm:0.74(3H,d,J=6.1 Hz),0.92(3H,d,J=5.9Hz),1.46(9H,d,J=1.2 Hz), 1.57(3H,dd,J=7.1,1.0Hz),1.82-1.91(1H,m),2.38(1H,brs),2.89(1H,dd,J=9.9,7.4Hz),3.11(1H,d,J=10.5 Hz),4.27-4.33(1H,m),5.02-5.05(1H,m),5.49(1H,q,J=7.0Hz),7.25-7.37(5H,m).

Referential Example 36(3S,4S)-3-(tert-Butoxycarbonylamino)-4-isopropyl-1-[(1R)-phenylethyl]pyrrolidine

A solution of boran.tetrahydrofuran complex in tetrahydrofuran (1.00mol/L, 63.9 mL, 63.9 mmol) was added dropwise to a solution of(3S,4S)-3-(tert-butoxycarbonylamino)-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine(7.38 g, 21.3 mmol) in tetrahydrofuran (150 mL) under cooling with ice,and the resultant mixture was stirred at room temperature for 15 hours.The reaction mixture was concentrated under reduced pressure.Subsequently, ethanol and water mixture solution (9:1) (176 mL) andtriethylamine (7 mL) were added to the residue, followed by refluxingfor 2 hours. The reaction system was concentrated under reducedpressure, and the residue was dissolved in chloroform (150 mL×2). Thesolution was washed by water (100 mL) and saturated brine (100 mL). Theobtained organic layer was dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. The obtained residue waspurified through silica gel column chromatography(chloroform:methanol=19:1), to thereby yield the title compound asyellow crystals (6.90 g, 97%).

¹H-NMR(400 MHz,CDCl3)δ ppm:0.76(3H,d,J=6.6 Hz),0.91(3H,d,J=6.4Hz),1.31(3H,d,J=6.6 Hz), 1.46(10H,s),1.73-1.83(1H,m),2.34(1H,t,J=9.4Hz),2.50(1H,t,J=9.7 Hz),2.67(1H,dd,J=9.2,4.0 Hz),2.79(1H,d,J=9.3Hz),3.37(1H,q,J=6.6 Hz),4.17-4.24(1H,m),4.92(1H,d,J=10.0Hz),7.20-7.34(5H,m).

Example 167-[(3S,4S)-3-Amino-4-isopropylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 16)

A 10% palladium carbon catalyst (M, water content: 52.8%, 5.00 g) wasadded to a solution of(3S,4S)-3-(tert-butoxycarbonylamino)-4-isopropyl-1-[(1R)-phenylethyl]pyrrolidine(5.68 g, 17.1 mmol) in ethanol (240 mL), and the resultant mixture wasstirred under a hydrogen gas atmosphere at 40° C. for 17 hours. Thecatalyst was removed through filtration (washing by ethanol), and thefiltrate was concentrated under reduced pressure. Subsequently, a 1mol/L aqueous sodium hydroxide solution (2 mL) was added to the residue,followed by extraction with chloroform (50 mL×5), and drying overanhydrous sodium sulfate. The solvent was removed under reducedpressure. To a solution of the obtained(3S,4S)-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-isopropylpyrrolidine(4.00 g, 17.1 mmol) sulfolane (20 mL), triethylamine (1.43 mL, 10.2mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid.difluoroboran complex (2.95 g, 8.54 mmol) were added, and theresultant mixture was stirred for 7 days. Thereafter, an ethanol-watermixture (9:1) (200 mL) and triethylamine (2 mL) were added to thereaction mixture, followed by refluxing for 30 minutes. The reactionsystem was concentrated under reduced pressure, and the residue wasdissolved in ethyl acetate (300 mL). The obtained solution was washed bya 10% aqueous citric acid solution (100 mL), water (100 mL×3), andsaturated brine (100 mL). The obtained organic layer was dried overanhydrous sodium sulfate, and the solvent was removed under reducedpressure. The obtained residue was purified through short silica gelcolumn chromatography (chloroform:methanol=49:1 to 9:1). Thethus-purified product was dissolved in concentrated hydrochloric acid(10 mL) under cooling with ice, and the resultant solution was stirredat room temperature for 15 minutes. Subsequently, the reaction mixturewas washed by chloroform (150 mL×3). The pH of the obtained aqueouslayer was adjusted to 11.0 with a 10 mol/L aqueous sodium hydroxidesolution under cooling with ice. The pH was adjusted again to 7.4 withhydrochloric acid, followed by extraction with chloroform (200 mL×4).The combined organic layer was dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure. The obtained residue wasrecrystallized from ethanol (in the presence of activated carbon), anddried under reduced pressure, to thereby yield the title compound aspale yellow crystals (1.32 g, 37%).

mp:124-127° C.

¹H-NMR(400 MHz,0.1N-NaOD)δ ppm:0.97(6H,dd,J=32.2,6.1Hz),1.16-1.28(1H,m),1.61(1H,dd,J=16.4,6.1Hz),1.69-1.78(1H,m),1.88-1.97(1H,m),2.43(3H,s),3.13(1H,d,J=10.3Hz),3.29(1H,t,J=8.3 Hz), 3.54(1H,s),3.68(1H,dd,J=17.5,7.7Hz),4.03-4.11(2H,m),5.02(1H,d,J=64.0 Hz),7.66(1H,d,J=14.2 Hz),8.42(1H,d,J=3.4 Hz).

Anal.:Calcd for C₂₁H₂₅F₂N₃O₃.1H₂O: C, 59.56; H, 6.43; F, 8.97; N, 9.92.Found: C, 59.38; H, 6.33; F, 9.06; N, 10.00.

IR(ATR):3444,2956,2929,2871,1728,1618,1545,1506,1468,1431,1358,1306,1230, cm⁻¹.

Referential Example 37(3S,4S)-3-(N-tert-Butoxycarbonyl-N-methyl)amino-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine

Sodium hydride (55%, 723 mg, 16.6 mmol) was added to a solution of(3S,4S)-3-(tert-butoxycarbonylamino)-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine(2.87 g, 8.28 mmol) in N,N-dimethylformamide (29 mL), and the resultantmixture was stirred at 0° C. for 10 minutes. Subsequently, methyl iodide(0.774 mL, 12.4 mmol) was added thereto at the same temperature,followed by stirring for 30 minutes. Thereafter, a saturated aqueousammonium chloride solution (50 mL) was added to the reaction mixture,followed by extraction with ethyl acetate (50 mL×2), and washing bywater (30 mL×2) and saturated brine (30 mL). The obtained organic layerwas dried over anhydrous sodium sulfate, and the solvent was removedunder reduced pressure. The obtained residue was purified through silicagel column chromatography (n-hexane:ethyl acetate=4:1 to 2:1), tothereby yield the title compound as colorless solid (2.59 g, 87%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.81-0.87(3H,m),0.96(3H,t,J=7.0Hz),1.46-1.48(10H,m),1.55(3H,d,J=7.4Hz),1.84-1.97(1H,m),2.75(3H,s),3.05(2H,dt,J=23.4,9.5 Hz),4.85(1H,d,J=8.8 Hz),5.57(1H,q,J=7.0 Hz),7.26-7.38(5H,m).

Referential Example 38(3S,4S)-3-(N-tert-Butoxycarbonyl-N-methyl)amino-4-isopropyl-1-[1-(R)-phenylethyl]pyrrolidine

A solution of boran.tetrahydrofuran complex in tetrahydrofuran (1.07mol/L, 21.5 mL, 23.0 mmol) was added dropwise under cooling with ice toa solution of(3S,4S)-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-isopropyl-2-oxo-1-[(1R)-phenylethyl]pyrrolidine(2.59 g, 7.28 mmol) in tetrahydrofuran (50 mL), and the resultantmixture was stirred at room temperature for 15 hours. The reactionmixture was concentrated under reduced pressure. Subsequently, anethanol and water mixture (9:1) (55 mL) and triethylamine (3 mL) wereadded to the residue, followed by refluxing for 2 hours. The reactionsystem was concentrated under reduced pressure, and the residue wasdissolved in chloroform (150 mL×2), followed by washing by water (100mL) and saturated brine (100 mL). The obtained organic layer was driedover anhydrous sodium sulfate, and the solvent was removed under reducedpressure. The obtained residue was purified through silica gel columnchromatography (chloroform:methanol=19:1), to thereby yield the titlecompound as yellow crystals (2.09 g, 84%).

¹H-NMR(400 MHz,CDCl₃)δ ppm:0.85(3H,dd,J=36.5,5.6 Hz),1.33(3H,d,J=5.9Hz),1.44(9H,s),1.86-2.00(1H,m),2.45(1H,t,J=9.9Hz),2.61-2.91(4H,m),3.01(3H,s),3.38(1H,q,J=6.5Hz),4.85(1H,brs),7.26(5H,t,J=13.0 Hz).

Referential Example 39(3S,4S)-3-(N-tert-Butoxycarbonyl-N-methyl)amino-4-isopropylpyrrolidine

(3S,4S)-3-(N-tert-Butoxycarbonyl-N-methyl)amino-4-isopropyl-1-[1-(R)-phenylethyl]pyrrolidine(1.39 g, 4.00 mmol) was dissolved in ethanol (56 mL). Subsequently, a10% palladium carbon catalyst (M, water content: 50.9%, 1.00 g) wasadded to the resultant solution, followed by stirring under hydrogenfloat at 40° C. for 12 hours. The catalyst was removed throughfiltration (washing by ethanol), and the filtrate was concentrated underreduced pressure. Thereafter, a 1 mol/L aqueous sodium hydroxidesolution (2 mL) was added to the residue. The resultant mixture wasextracted with chloroform (50 mL×5), followed by drying over anhydroussodium sulfate. The solvent was removed under reduced pressure, tothereby yield the unpurified title compound as colorless syrup (942 mg,97%).

¹H-NMR(400 MHz,CDCl₃)δppm:1.47(9H,s),2.54-2.68(1H,m),2.85(3H,s),2.97(1H,dd,J=11.3,7.1Hz),3.07(1H,dd,J=11.5,5.6 Hz),3.16-3.23(2H,m),4.36(1H,ddd,J=47.6,9.3,6.4Hz), 4.48(1H,ddd,J=46.8,9.1,5.1 Hz),4.41-4.48(1H,m).

Example 176-Fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-7-[(3S,4S)-3-isopropyl-4-methylaminopyrrolidin-1-yl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (Compound No. 17)

Triethylamine (0.335 mL, 2.40 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid.difluoroboran complex (690 mg, 2.00 mmol) were added to a solutionof(3S,4S)-3-(N-tert-butoxycarbonyl-N-methyl)amino-4-isopropylpyrrolidine(942 mg, 3.89 mmol) in sulfolane (5 mL), and the resultant mixture wasstirred at room temperature for 12 days. Subsequently, cold water (100mL) was added to the reaction mixture, and a precipitated solid wascollected through filtration. Thereafter, an ethanol and water mixture(9:1) (100 mL) and triethylamine (1 mL) were added to the obtainedsolid, followed by refluxing for 30 minutes. The reaction system wasconcentrated under reduced pressure, and the residue was dissolved inethyl acetate (150 mL×2). The solution was washed by a 10% aqueouscitric acid solution (100 mL), water (100 mL×2), and saturated brine(100 mL). The obtained organic layer was dried over anhydrous sodiumsulfate, and the solvent was removed under reduced pressure. Theobtained residue was purified through short silica gel columnchromatography (chloroform:methanol=19:1 to 4:1). The purified productwas dissolved under cooling with ice in concentrated hydrochloric acid(5 mL), followed by stirring at room temperature for 30 minutes.Thereafter, the reaction mixture was washed by chloroform (50 mL×3). ThepH of the obtained aqueous layer was adjusted to 12.0 with a 10 mol/Laqueous sodium hydroxide solution under cooling with ice. The pH wasadjusted again to 7.4 with hydrochloric acid. The resultant mixture wasextracted with chloroform (100 mL×3). The combined organic layer wasdried over anhydrous sodium sulfate, and the solvent was removed underreduced pressure. The obtained residue was purified through preparativechromatography, recrystallized from ethanol-diethyl ether, and driedunder reduced pressure, to thereby yield the title compound as yellowcrystals (49.0 mg, 6%).

mp:168-171° C.

¹H-NMR(400 MHz,0.1N-NaOD)δppm:1.26-1.38(1H,m),1.58-1.69(1H,m),2.36(3H,s),2.54(3H,s),2.82-2.93(1H,m),3.41(1H,q,J=5.0Hz),3.49(1H,q,J=5.8 Hz),3.58(2H,d,J=6.9 Hz),3.79(1H,ddd,J=9.6,6.1,1.5Hz),4.12(1H,dt,J=8.6,5.4 Hz),4.72-4.80(2H,m),5.00(1H,d,J=65.0 Hz),7.70(1H,d,J=14.0 Hz),8.48(1H,d,J=2.7 Hz).

Anal.:Calcd for C₂₂H₂₇F₂N₃O₃.0.75H₂O: C, 61.03; H, 6.63; F, 8.78; N,9.70. Found: C, 60.68; H, 6.14; F, 9.04; N, 9.59.

IR(ATR):3082,2960,2935,2870,2796,1716,1614,1508,1466,1431,1360,1335,1313,1257,1225 cm⁻¹.

Test Example 1 Antibacterial Activity

The antimicrobial activities of the compounds of the present inventionwere determined according to a standard method designated by the JapanSociety of Chemotherapy. The results are expressed by MIC (μg/mL) (Table1). In Table 1, S. aureus 870307 and S. pneumonia J24 arequinolone-resistant bacteria.

TABLE 1 Compound Compound Compound Compound Compound Compound CompoundBacterium No. 1 No. 2 No. 3 No. 5 No. 8 No. 16 E. coli NIHJ 0.006 ≦0.0030.006 0.006 0.006 0.006 S. flexneri 2A 5503 0.006 ≦0.003 0.006 0.0060.025 0.006 P. Vulgalis 08601 0.012 0.006 0.006 0.012 0.025 0.025 K.pneumoniae TYPE I 0.025 0.025 0.05 0.025 0.05 0.025 S. marcescens 101000.1 0.025 0.025 0.05 0.1 0.05 P. aeruginosa 32104 0.2 0.05 0.1 0.2 0.20.2 P. aeruginosa 32121 0.1 0.025 0.05 0.1 0.1 0.1 S. maltophilia IID1275 0.05 0.05 0.2 0.05 0.2 0.05 S. aureus FDA 209P 0.006 0.006 0.0120.006 0.012 0.006 S. epidermidis 56500 0.05 0.025 0.05 0.025 0.1 0.025S. pyogenes G-36 0.05 0.025 0.1 0.025 0.1 0.025 E. faevcalis ATCC 194330.1 0.1 0.1 0.1 0.1 0.1 S. aureus 870307 0.2 0.39 0.78 0.2 0.39 0.1 S.pneumoniae J24 0.025 0.025 0.1 0.025 0.1 0.05 Compound ComparativeComparative Comparative Bacterium Compound A Compound B Compound C LVFXCPFX MFLX E. coli NIHJ 0.006 0.012 0.012 0.012 ≦0.003 0.006 S. flexneri2A 5503 0.006 0.012 0.025 0.025 0.006 0.012 P. Vulgalis 08601 0.0060.006 0.05 0.012 ≦0.003 0.025 K. pneumoniae TYPE I 0.05 0.05 0.05 0.10.025 0.05 S. marcescens 10100 0.05 0.05 0.2 0.1 0.025 0.1 P. aeruginosa32104 0.1 0.1 0.39 0.2 0.05 0.39 P. aeruginosa 32121 0.05 0.025 0.2 0.10.025 0.2 S. maltophilia IID 1275 0.1 0.39 0.2 0.39 0.78 0.1 S. aureusFDA 209P 0.012 0.012 0.025 0.2 0.1 0.025 S. epidermidis 56500 0.05 0.10.1 0.39 0.2 0.1 S. pyogenes G-36 0.05 0.1 0.2 0.78 1.56 0.2 E.faevcalis ATCC 19433 0.1 0.2 0.2 0.78 0.78 0.2 S. aureus 870307 0.393.13 1.56 >6.25 >6.25 0.78 S. pneumoniae J24 0.025 0.1 0.1 0.78 0.390.025

As is clear from Table 1, the compound of the present inventionexhibited antibacterial activity against a broad range of bothGram-positive bacteria, including quinolone-resitant bacteria, andGram-negative bacteria, and the activity was found to be almostcamparable to that of any of the known synthetic quinolone antibacterialagents.

Test Example 2 Effect of Inducing Convulsion

(1) Sample to be Tested and Administration

The compounds (Compound Nos. 1, 2, and 3) of the present invention andComparative Compounds A and B were individually dissolved in 5% glucose,to thereby prepare several samples to be tested. Groups of six-week-oldSlc:ddY male mice (body weight: 23.9 to 27.4 g), each group consistingof 6 mice, were employed in the test. Each of the above-prepared sampleswas administered intracisternally to each mouse belonging to each groupat concentrations shown in Tables 2 and 3 (5 μL/mouse). During 30minutes after administration, it was observed whether each mousesuffered convulsion or died in an individual cage. Administration wasperformed according to the method described by Ueda, et al. (Reference1).

Reference 1: Ueda H, Amano H, Shiomi H, Takagi H: Comparison of theanalgesic effects of various opioid peptides by a newly devisedintracisternal injection technique in conscious mice. Eur J Pharmacol.1979; 56: 265-8.

(2) Statistical Analysis

Significant difference test between groups was performed in terms ofincidence of convulsion according to the Fisher's direct probabilitycalculation method (one-sided test, level of significance: P<0.05). Onthe basis of percentile incidence of convulsion, CD₅₀ (50% concetrationfor inducing convulsion) and 95% confidence limit were calculated by theProbit method. The results are shown in Tables 2, 3, and 4.

TABLE 2 Dose Effect of inducing convulsion (μg/5 μL/mouse, i.cist.)Compound No. 1 Comp. Compound A 0 0/6 0/6 3.125 — 0/6 6.25 — 1/6 12.50/6 3/6 25 0/6    5/6**^(,##) 37.5 0/6 — 50  4/6* — CD₅₀ 49.2^(b)) 12.7(7.7-24.9)^(a)) Control: 5% glucose i.cist.: Intracisternallyadministration *P < 0.05, **P < 0.01: significant difference betweenincidence of convulsion of test group and that of control group(calculated by the Fisher's direct probability calculation method)^(##)P < 0.01: significant difference between incidence of convulsion ofgroup employing Compound No. 1 and that of each of the other groups^(a))95% confidence limit ^(b))95% confidence limit could not beanalyzed CD₅₀: 50% concetration for inducing convulsion —: Not testable

TABLE 3 Dose Effect of inducing convulsion (μg/5 μL/mouse, i.cist.)Compound No. 3 Comp. Compound B 0 0/6 0/6 12.5 — 0/6 25 0/6 3/6 50 1/6   5/6**^(,#) 75  4/6* — 100  6/6** — CD₅₀ 64.5 (47.7-80.8) 28.6(16.8-48.5)^(a))

TABLE 4 Dose Effect of inducing convulsion (μg/5 μL/mouse, i.cist.)Compound No. 2 0 0/6 5 0/6 15 5/6 50 6/6

As is clear from Tables 2 to 4, the compound of the present inventionwas found to have low effect of inducing convulsion.

Test Example 3 Effect of Inducing Chromosomal Aberration

Preparation of Specimen

Compound Nos. 1, 2, and 3 of the present invention, and ComparativeCompounds C, D, and E were individually dissolved in 0.1 mol/L NaOH, tothereby prepare six sample solutions. From each sample solution, samplesto be tested having a concentration of 1, 3, 10, 30, 50, and 100 μg/mL(as reduced to free form) were prepared. As a negative control, 0.1mol/L NaOH was employed.

Chinese hamster lung (CHL/IU) cells in logarithmic growth phase wereemployed. The cells were treated in a culture solution containing eachsample to be tested for 24 hours. Subsequently, a chromosome specimenwas prepared from the thus-treated cells. The culture solution employedwas an Eagle's MEM (product of NISSUI PHARMACEUTICAL CO., LTD.) mediumcontaining 15% fetal bovine serum (15% FBS-MEM).

Observation of Chromosomal Aberration

In each sample concetration, 100 cells during metaphase were observed,to thereby record types and numbers of the chromosomal aberration.

Statistical Analysis

Incidence of cells exhibiting chromosomal aberration was determinedthrough analysing difference between incidence of each negative controlgroup and incidence of each sample-treated group by χ²-test with Yatescorrection at a one-side significance level of 5%. The percentage of theresults without gap (TA) (percentile induction of structural aberrationTA %) was shown in Table 5.

Reference 1: “Atlas of chromosome aberration by chemicals” (AsakuraPublishing Co., Ltd. 1998), edited by Japanese Environmental MutagenSociety•MMS study group

Reference 2: Iyakusingikai No. 1604, Guideline for Genotoxicity Test

TABLE 5 Percentile induction of structural aberration (TA %)Concentration Compound Compound Compound Comp. Comp. Comp. (μg/mL) No. 1No. 2 No. 3 Compound E Compound C Compound D Negative control 2 4 2 4 44 1 0 1 3 0 3 4 3 1 1 0 3 4 0 10 2 1 2 1 5 3 30 3 2 1 6 72* 63* 50 2 3 5TOX*¹ 98* 98* 100 7 8 9 TOX*¹ TOX*² 100*  *Significant differencebetween incidence of cells exhibiting chromosomal aberration of testgroup and that of nagative control (calculated by χ²-test with Yatescorrection, p < 0.05) *¹C-mitosis was observed *²No metaphase wasobserved

Comparative Compound E5-amino-7-[(3S,4S)-3-amino-4-ethyl-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylicacid (see Patent Document 9)

As is clear from Table 5, in the case where Compound Nos. 1, 2, and 3 ofthe present invention were employed, significant increase in number ofthe cells exhibiting chromosomal aberration was not observed at aconcetration of up to 100 μg/mL. In the chromosomal aberration test, acompound causing chromosomal aberration in 10% or less cells isevaluated negative.

In the case of Comparative Compound E, significant increase in number ofthe cells exhibiting chromosomal aberration was not observed at aconcetration of up to 30 μg/mL. However, at a concentration of 50 μg/mLor more, C-mitosis, which may be caused by inhibition of cell division,and ski-pair-like chromosomes are observed considerably, resulting infail to be evaluated. Comparative Compounds C and D exhibited effects ofinducing chromosomal aberration at a concentration of 30 μg/mL or more.Particularly, in the case of Comparative Compound C, chromosomes couldnot be observed at a concentration of 100 μg/mL due to its cytotoxicity.

We claim:
 1. A compound of formula (I):

wherein R¹ represents a hydrogen atom, a C₁-C₆ alkyl group, or asubstituted carbonyl group derived from an amino acid, a dipeptide, or atripeptide; R² represents a hydrogen atom or a C₁-C₆ alkyl group, and,when either one or each of R¹ and R² is an alkyl group, the alkyl groupmay be substituted with a group selected from the group consisting of ahydroxyl group, an amino group, a halogen atom, a C₁-C₆ alkylthio group,and a C₁-C₆ alkoxy group; R³ represents a C₂-C₆ alkyl group, a C₂-C₆alkenyl group, or a C₃-C₆ cycloalkyl group; R⁴ and R⁵ each independentlyrepresents a hydrogen atom or a C₁-C₆ alkyl group; R⁷ represents ahydrogen atom, a phenyl group, an acetoxymethyl group, apivaloyloxymethyl group, an ethoxycarbonyl group, a choline group, adimethylaminoethyl group, a 5-indanyl group, a phthalidyl group, a5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl group, a 3-acetoxy-2-oxobutyl group,a C₁-C₆ alkyl group, a C₂-C₇ alkoxymethyl group, or a phenylalkyl groupformed of a C₁-C₆ alkylene group and a phenyl group; X¹ represents ahydrogen atom or a halogen atom; and A represents a partial structurerepresented by the following formula (II):

wherein X² and R⁶ together form a cyclic structure so as to contain apart of the quinolone nucleus to form a pyridobenzoxazine skeleton ofthe formula:

wherein the arrow denotes a bond connected with the pyrrolidine ring offormula (I); or a salt thereof.
 2. The compound as described in claim 1or a salt thereof, wherein the compound of formula (I) is a compoundrepresented by the following formula:

wherein R¹, R², R³, R⁴, R⁵, R⁷, and X¹ are as defined in claim
 1. 3. Thecompound as described in claim 1 or a salt thereof, wherein each of R¹and R² in formula (I) is a hydrogen atom.
 4. The compound as describedin claim 1 or a salt thereof, wherein one of R¹ and R² in formula (I) isa hydrogen atom, and the other is a methyl group.
 5. The compound asdescribed in claim 1 or a salt thereof, wherein R³ in formula (I) is anethyl group, an n-propyl group, a vinyl group, an isopropyl group, or acyclopropyl group.
 6. The compound as described in claim 1 or a saltthereof, wherein R³ in formula (I) is an ethyl group.
 7. The compound asdescribed in claim 1 or a salt thereof, wherein each of R⁴ and R⁵ informula (I) is a hydrogen atom.
 8. The compound as described in claim 1or a salt thereof, wherein X¹ in formula (I) is a fluorine atom.
 9. Thecompound as described in claim 1 or a salt thereof, wherein R⁷ informula (I) is a hydrogen atom.
 10. The compound as described in claim 1or a salt thereof, wherein a compound of formula (I) is astereochemically single compound. 11.(3S)-10-[(3S,4S)-3-Amino-4-ethylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid or a salt thereof.
 12. A drugcontaining, as an active ingredient, the compound as recited in claim 1or a salt thereof.
 13. An antibacterial drug containing, as an activeingredient, the compound as recited in claim 1 or a salt thereof.
 14. Aninfectious disease treating drug containing, as an active ingredient,the compound as recited in claim 1 or a salt thereof.
 15. A method forproducing a drug, comprising incorporating the compound as recited inclaim 1 or a salt thereof into a tablet, a powder, a granule, a capsule,a solution, a syrup, an elixir, an oil, or an aqueous suspension.
 16. Amethod for producing an antibacterial drug, comprising incorporating thecompound as recited in claim 1 or a salt thereof into a tablet, apowder, a granule, a capsule, a solution, a syrup, an elixir, an oil, oran aqueous suspension.
 17. A method for producing an infectious diseasetreating drug, comprising incorporating the compound as recited in claim1 or a salt thereof into a tablet, a powder, a granule, a capsule, asolution, a syrup, an elixir, an oil, or an aqueous suspension.